Molecular Marker-Assisted Pyramiding of SCM3, Wx, and BADH2 Genes for the Development of High-Yield, Superior-Quality, and Lodging-Resistant Rice Varieties

Molecular Marker-Assisted Pyramiding of SCM3, Wx, and BADH2 Genes for the Development of High-Yield, Superior-Quality, and Lodging-Resistant Rice Varieties | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Molecular Marker-Assisted Pyramiding of SCM3, Wx, and BADH2 Genes for the Development of High-Yield, Superior-Quality, and Lodging-Resistant Rice Varieties Jinlong Hu, Zexu Zhou, Yu Zhang, Pan Mao, Xin Zhao, Nianbing Zhou, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6845239/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Utilizing the functional divergence sites of SCM3 , we established a molecular marker and performed SCM3 genotyping along with an evaluation of lodging resistance traits in 78 rice germplasm resources. Fourteen accessions with the SCM3⁹³¹¹ genotype demonstrated exceptional lodging resistance. In particular, Yangchan 93033 and Yangchannuo 1 exhibited strong stems, high bending resistance, and a significant number of grains per panicle, indicating their potential as elite donor parents for breeding lodging-resistant rice. Resequencing analysis revealed a rare variant site in the SCM3 gene of both Yangchan 93033 and Yangchannuo 1, and the derived KASP marker SCM3_k_28430214 was confirmed as an effective tool for molecular marker-assisted selection. A hybrid population was created by crossing Yangchan 93033 with Wuxiangjing 5245, a high-quality japonica cultivar known for its superior taste, to form a segregating breeding population. KASP markers targeting SCM3 , Wx , and BADH2 were utilized for genotypic screening of the progeny. Among 44 F₆ stable lines carrying SCM3⁹³¹¹ , there was a significant enhancement in lodging resistance compared to Wuxiangjing 5245, validating the effective selection capability of the SCM3_k_28430214 marker in improving lodging resistance. Through molecular marker-assisted selection, we rapidly developed a new breeding line, 24HD134, which integrates favorable alleles of SCM3 , Wx , and BADH2 . This line demonstrates high yield potential, superior grain quality, and improved lodging resistance, offering valuable genetic resources for rice breeding programs. MAS SCM3 gene pyramiding rice lodging resistance rice yield rice quality Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Rice is a crucial staple crop in China, with its high and stable yield being essential for national food security. Lodging, which can occur during the mid to late growth stages, is influenced by environmental conditions, cultivation practices, and the genetic traits of the rice varieties. Mild lodging causes the plants to lean, resulting in reduced light penetration within the canopy and a decline in photosynthetic efficiency. In severe cases, broken stems hinder nutrient transport and water absorption, hastening the aging of the rice plants. As a result, lodging leads to inadequate grain filling and a substantial decrease in yield (Guo et al., 2020 ). Furthermore, when rice panicles are positioned close to the ground, it can result in grain mold or pre-harvest sprouting, negatively impacting rice quality and reducing seed germination rates, while also increasing harvesting costs (Mullangie et al., 2024 ). Lodging in rice is primarily influenced by four key factors. Firstly, adverse environmental conditions such as heavy rainfall and strong winds can cause rice plants to lean or even break at the stem. Secondly, varietal traits play a significant role; tall varieties with large panicles often possess a higher center of gravity, which compromises their resistance to lodging (Merugumala et al., 2019 ). Additionally, varieties characterized by thin and soft stems, elongated basal internodes, wide tillering angles, loose plant structures, and shallow root systems tend to have diminished lodging resistance (Mullangie et al., 2024 ). Thirdly, cultivation management practices are critical; excessive nitrogen fertilizer application can lead to increased plant height and elongated basal internodes, while simultaneously reducing stem wall thickness and diameter, thereby weakening the lodging resistance of rice (Zhang et al., 2016 ). High planting densities may also result in taller plants with thinner, weaker stems, as well as decreased dry weight and mechanical strength per internode, further impairing the plant's lodging resistance. Lastly, planting methods can impact lodging; simplified cultivation techniques, such as direct seeding, may lead to uneven sowing and shallow seed placement, resulting in overcrowding and shallow root systems, which further exacerbate lodging issues (Yadav et al., 2017 ). The development and cultivation of lodging-resistant rice varieties are recognized as economically viable strategies to mitigate the issue of rice lodging. The resistance of rice to lodging is primarily influenced by characteristics such as plant height, stem diameter, stem strength, and tillering angle (Sowadan et al., 2018 ; Mulsanti et al., 2018 ; Guo et al., 2020 ; Mullangie et al., 2024 ). A negative correlation exists between plant height and lodging resistance; taller plants possess a higher center of gravity, rendering them more prone to lodging. The stem serves as the principal support structure for rice, and varieties with thicker stems typically demonstrate enhanced lodging resistance (Samadi et al., 2019 ; Mullangie et al., 2024 ). In the 1960s, the introduction of semi-dwarf genes effectively addressed the lodging issue in rice (Asano et al., 2007 ). Nevertheless, the biomass accumulation of semi-dwarf rice varieties is constrained by their reduced height, which limits further yield improvements. Consequently, a strategic increase in plant height can boost the biological yield of rice, while enhancing stem thickness and strength can improve lodging resistance. This strategy facilitates an increase in yield potential while simultaneously mitigating the risk of lodging. To date, several quantitative trait loci (QTLs) associated with stem thickness and strength in rice have been successfully cloned, including SCM2 , SCM3 / OsTB1 , and IPA1 , among others. SCM2 encodes an F-box protein predominantly expressed in the apical meristem and lateral organ primordia (Ookawa et al., 2010 ). Enhanced expression of SCM2 results in increased stem diameter, improved mechanical strength, and a notable rise in the number of grains per panicle (Ookawa et al., 2010 ). SCM3 encodes a transcription factor from the TCP family and functions as a negative regulator of tiller number in rice. The SCM3 allele found in indica varieties, such as 9311 and Chugoku 117, possesses a 4-bp insertion in the 5' UTR region, leading to elevated expression levels that contribute to greater stem thickness and strength. In contrast, japonica varieties like Nipponbare and Koshihikari lack this 4-bp insertion, resulting in lower SCM3 expression and thinner stems (Yano et al., 2015 ). By employing CRISPR/Cas9 gene editing technology to modify the promoter region of SCM3 , researchers have developed favorable allelic variants with increased gene expression, which in turn enhances stem thickness and the number of grains per panicle (Cui et al., 2020 ). The ideal plant architecture gene IPA1 encodes a Squamosa promoter-binding protein-like transcription factor, OsSPL14, which directly binds to the promoter of SCM3 to inhibit tiller formation (Zhang et al., 2017 ; Wang et al., 2021 ). Gene editing of the IPA1 promoter has led to the creation of novel allelic variants with increased expression levels, resulting in simultaneous enhancements in panicle weight and grain number, as well as thicker stems and roots (Song et al., 2022 ). Loss of function of Gn1a/OsCKX2 leads to the accumulation of cytokinins in the crown root tips, accelerating the development of adventitious roots, increasing the number of grains per panicle in rice plants, and exhibiting excellent lodging resistance (Tu et al., 2022; J. Zhang et al., 2024). Recent research has successfully cloned the STRONG2 gene, known for its high yield and resistance to lodging, through genome-wide association studies (GWAS). This gene encodes a cellulose synthase-like enzyme (CSLA) that plays a crucial role in the development of the secondary cell wall by modulating the composition of the cell wall, which in turn enhances stem thickness and strength (Zhao et al., 2025 ). Furthermore, the analysis indicated that the beneficial allele of STRONG2 is predominantly found in improved indica rice varieties, whereas its occurrence is comparatively lower in enhanced japonica rice varieties (Zhao et al., 2025 ). Consequently, the STRONG2 gene presents significant potential for future genetic enhancement and breeding strategies aimed at improving lodging resistance in japonica rice. The BADH2 gene encodes the enzyme betaine aldehyde dehydrogenase, which facilitates the conversion of γ-aminobutyraldehyde (GABald) into γ-aminobutyric acid (GABA). This process is integral to plant stress responses and metabolic regulation (Li et al., 2024 ; Li et al., 2024 ). Mutations that result in the loss of function of BADH2 lead to an accumulation of GABald, which is a precursor in the biosynthesis of 2-acetyl-1-pyrroline (2AP). This accumulation subsequently promotes the increased presence of the aromatic compound 2AP in rice grains (Hui et al., 2021 ; Chen et al., 2024 ). Therefore, mutations that inactivate BADH2 represent a significant target for molecular breeding strategies aimed at enhancing the fragrance of rice. The Wx gene encodes granule-bound starch synthase I (GBSSI), which is responsible for catalyzing the synthesis of amylose during the development of the endosperm. The activity of the Wx gene is a determining factor for the amylose content in rice, which in turn affects important eating quality characteristics such as stickiness, hardness, and retrogradation of cooked rice (Zhang et al., 2019 ; Huang et al., 2020 ). Variations in the Wx gene can lead to changes in GBSSI activity, resulting in different allelic forms that significantly influence the eating quality of rice (Huang et al., 2020 ; Zeng et al., 2020 ). The Wx mp allele, which is defined by a G to A mutation in the fourth exon (vg0601767613), causes a partial reduction in GBSSI activity, leading to an intermediate amylose content that ranges from 5–12% (Huang et al., 2020 ; Zhou et al., 2020 ). As a result, the Wx mp allele has been instrumental in improving the eating quality of japonica rice in Jiangsu Province. Molecular markers derived from functional variation sites in genes facilitate the swift and effective identification of genotypes linked to desired traits in breeding materials. This approach minimizes the effort required for phenotypic assessment of these traits and enhances the efficiency of selection processes (Kim et al., 2016; Ueda et al., 2025 ). Jiangsu Province represents the largest area for japonica rice cultivation in southern China. However, in recent years, extensive lodging in rice production has frequently resulted in decreased yields and compromised quality. The incidence and intensity of rice lodging have notably escalated, largely due to extreme weather events, posing a significant threat to the stability and high productivity of rice cultivation. Incorporating enhanced allelic variations that improve stem strength into traditional japonica rice cultivars in the middle and lower reaches of the Yangtze River represents a promising strategy for developing rice varieties resistant to lodging. We evaluated 78 japonica rice germplasm resources from Jiangsu Province for their lodging resistance and yield-related traits, identifying 14 accessions that possess the advantageous SCM3 allele. Notably, Yangchan 93033 and Yangchannuo 1 demonstrated strong stems and high yields, positioning them as valuable germplasm for the development of high-yielding, lodging-resistant rice varieties. To enhance quality traits, we performed a cross between Yangchan 93033 and Wuxiangjing 5245, a premium japonica rice variety known for its favorable alleles associated with fragrance and eating quality. Through multi-generational molecular marker-assisted selection (MAS) and field-based agronomic trait selection, we successfully generated novel japonica rice materials characterized by high yield, lodging resistance, and exceptional eating quality. Results Creation of Indel Molecular Markers for Functionally Distinct Regions in SCM3 Previous research has established that a 4-bp insertion in the 5' untranslated region (UTR) of the SCM3 gene enhances its expression, leading to an increase in stem diameter (Yano et al., 2015 ). This 4-bp variation is therefore recognized as a functional differential site within the SCM3 gene. An analysis using RiceVarMap v2.0 ( https://ricevarmap.ncpgr.cn/ ) indicated that 97.2% of indica rice varieties contain the 4-bp insertion at the specific locus vg0328428721, whereas only 57.3% of japonica rice varieties exhibit the same insertion. This suggests that the advantageous allele of SCM3 is relatively less common in japonica rice. Thus, promoting the use of this allele in future breeding initiatives could significantly enhance lodging resistance in japonica rice. According to RiceVarMap v2.0, the variation site vg0328428721, situated in the promoter region of the SCM3 gene, displays a 4-bp insertion in the 9311 variety, while this insertion is absent in the Nipponbare (NPB) variety (Fig. S1 A). A pair of primers, SCM3_indel_2F/R (Table S1 ), was designed to flank the variation site vg0328428721. PCR amplification was conducted using genomic DNA from the 9311 and Nipponbare (NPB) varieties as templates. The PCR products were subsequently analyzed through electrophoresis on an 8% polyacrylamide gel. The results revealed distinct target bands of 118 bp and 114 bp for the 9311 and NPB varieties, respectively (Fig. S1 B). This confirms the presence of the 4-bp insertion in the 9311 variety and its absence in NPB, establishing a reliable molecular marker for differentiating between these two genotypes at the SCM3 locus. Consequently, the two alleles were designated as SCM3 9311 and SCM3 NPB , respectively. A. The gene structure of SCM3 and the positions of functional variation sites. B. Polyacrylamide gel electrophoresis analysis of SCM3_indel_2. Identification of Characteristics for Lodging Resistance and Genotypic Analysis of the SCM3 Gene in 78 Japonica Rice Varieties A total of 78 rice varieties and advanced lines, either recently approved or currently under evaluation in Jiangsu Province, were randomly selected for allelic genotyping of the SCM3 gene using the SCM3indel-2 molecular marker. The analysis revealed that 14 rice varieties carried the SCM3 9311 allele, while the remaining 64 varieties exhibited the SCM3 NPB allele (Table S2 ). To evaluate the influence of different SCM3 alleles on rice lodging resistance, we investigated six key traits associated with lodging resistance in these 78 rice materials. These traits included plant height (PH), bending strength (BS), lodging index (LI), length of the second internode from the plant root (IL2), culm outer diameter at the cross-section of the second internode from the plant root (CD2), and culm thickness at the cross-section of the second internode from the plant root (CT2) (Table S2 ). A comparative analysis was conducted to evaluate the trait differences between the SCM3 9311 and SCM3 NPB genotypes (Table S3). The average culm diameter (CD2) for rice varieties harboring the SCM3 9311 allele was recorded at 5.57 mm, while those with the SCM3 NPB allele averaged 5.35 mm, indicating a statistically significant difference (Fig. 1 A). Furthermore, rice varieties with the SCM3 9311 allele demonstrated an average stem wall thickness of 0.84 mm at CT2, in contrast to 0.80 mm for varieties with the SCM3 NPB allele, which also exhibited a significant difference (Fig. 1 B). The average breaking strength (BS) for rice varieties containing the SCM3 9311 allele was 21.61 N, significantly exceeding the 18.56 N observed in varieties with the SCM3 NPB allele (Fig. 1 C). Regarding internode length (IL2), rice varieties with the SCM3 9311 allele averaged 8.27 mm, which was significantly shorter than the 8.71 mm recorded for those with the SCM3 NPB allele (Fig. 1 D). However, no significant difference in plant height (PH) was observed between the two genotypes (Fig. 1 E). Lastly, the lodging index (LI) for rice varieties possessing the SCM3 9311 allele was significantly lower at 40.05 compared to 43.43 for those with the SCM3 NPB allele (Fig. 1 F). These results indicate that the SCM3 9311 allele enhances stem strength and increases resistance to lodging in rice. To thoroughly assess the breeding potential of rice germplasm possessing the SCM3 9311 allele, we performed a statistical analysis on several traits across 78 rice accessions. These traits included the number of grains per panicle (NGP), seed setting rate (SP), thousand grain weight (TGW), grain length (GL), grain width (GW), number of effective panicles (PN), and yield per plant (YP). The analysis revealed that the average NGP for rice germplasm with the SCM3 9311 allele was 212.18, which slightly exceeded the average of 199.75 for germplasm with the SCM3 NPB allele (Fig. 2 A). Additionally, the average TGW for the SCM3 9311 allele was 26.30 g, marginally higher than the 25.85 g observed in the SCM3 NPB allele (Fig. 2 B). The average GL for germplasm carrying the SCM3 9311 allele was 7.98 mm, significantly greater than the 7.74 mm for the SCM3 NPB allele (Fig. 2 C). However, no significant differences were found in SP, GW, and PN between the two alleles (Fig. 2 D- 2 E). Furthermore, the average yield per plant for germplasm with the SCM3 9311 allele was 33.49 g, which was significantly higher than the 31.63 g for those with the SCM3 NPB allele (Fig. 2 F). These findings suggest that rice germplasm carrying the SCM3 9311 allele demonstrates enhanced lodging resistance and yield characteristics. Consequently, through molecular marker-assisted selection, the favorable SCM3 9311 allele can be incorporated into japonica rice varieties with desirable traits to develop high-yield, high-quality, lodging-resistant rice cultivars. A comparative analysis was conducted on various parameters, including the diameter of the second basal internode (A), wall thickness of the second basal internode (B), stem breaking resistance (C), length of the second basal internode (D), plant height (E), and lodging index (F) between the SCM3 9311 and SCM3 NPB genotypes across 78 japonica rice materials. Welch's Two Sample t -test was employed to assess the significance of the observed differences. A comparative analysis was conducted on various traits, including the number of grains per panicle (A), 1000-grain weight (B), grain length (C), grain width (D), plant height (E), and yield per plant (F), between the SCM3 9311 and SCM3 NPB genotypes across 78 japonica rice materials. Welch's Two Sample t -test was employed to assess the significance of the observed differences. Elite germplasm resources were chosen as parental lines to develop rice varieties characterized by high yield, exceptional quality, and resistance to lodging Among the 14 rice germplasm materials possessing the SCM3 9311 allele, Yangchan 93033 and Yangchannuo 1 demonstrated superior overall performance in terms of F, CD2, TGW, YP, and NGP (Fig. S2 ). Despite all 14 rice germplasm accessions carrying the advantageous SCM3 9311 allele, notable differences in lodging resistance traits were observed among them. This phenotypic variation is likely due to differences in their genetic backgrounds, which may affect the expression or efficacy of the SCM3 9311 allele in providing lodging resistance. A comparative analysis was conducted on various agronomic traits of 14 japonica rice materials possessing the SCM3 9311 genotype. The traits examined included the number of grains per panicle (A), 1000-grain weight (B), grain length (C), grain width (D), plant height (E), and yield per plant (F). For the multiple comparison analysis, a significance level of LSD_0.01 was employed. Wuxiangjing 5245 is recognized as a high-quality aromatic soft rice variety; however, it exhibits lower yield and lodging resistance compared to Yangchan 93033 (Fig. S3). The critical dimensions (CD2) and breaking strength (BS) of Yangchan 93033 were significantly higher than those of Wuxiangjing 5245, indicating its superior lodging resistance (Fig. S3A and S3C). Furthermore, Yangchan 93033 demonstrated markedly elevated net grain production (NGP), thousand grain weight (TGW), and yield potential (YP) relative to Wuxiangjing 5245, highlighting its considerable yield enhancement potential (Fig. S3A and S3C). Therefore, to create new rice varieties that combine high yield, exceptional quality, and improved lodging resistance, we developed hybrid combinations by crossing Yangchan 93033 and Yangchannuo 1 (which serve as donors of the SCM3 gene) with the high-quality and palatable japonica rice variety Wuxiangjing 5245 (Fig. S3G, S3H, and S3L). A comparative analysis was conducted on various parameters between two japonica rice varieties, Yangchan 93033 and Wuxiangjing 5245. The parameters examined included the diameter of the second basal internode (A), wall thickness of the second basal internode (B), stem breaking resistance (C), length of the second basal internode (D), plant height (E), lodging index (F), number of grains per panicle (G), 1000-grain weight (H), grain length (I), grain width (J), number of productive panicles (K), and yield per plant (L). Welch's Two Sample t -test was employed to assess the significance of the differences observed. Further examination of the basal stems of Yangchan 93033 and Wuxiangjing 5245 revealed that the vascular bundle size in Yangchan 93033 was considerably larger than that in Wuxiangjing 5245 (Fig. 3 ). Cross-sectional microscope images of the second internode at the base for Yangchan 93033 (A) and Wuxiangjing 5245 (B). Cross-sectional paraffin section microscope images of the second internode at the base for Yangchan 93033 (C) and Wuxiangjing 5245 (D). Validation of the SCM3 Locus for Lodging Resistance Through Bulked Segregant Analysis (BSA). To confirm that the SCM3 gene is the primary gene associated with lodging resistance in Yangchannuo 1, we measured the diameter of the second internode at the base of the main stem panicle in 200 individual plants from the F2 population derived from the cross between Yangchannuo 1 and Wuxiangjing 5245. We subsequently selected and pooled leaves from 30 plants exhibiting diameters similar to those of Yangchannuo 1 and 30 plants resembling Wuxiangjing 5245 for resequencing analysis. Both parental lines, Yangchannuo 1 and Wuxiangjing 5245, were also included in the resequencing as controls. Bulked segregant analysis (BSA) identified a significant quantitative trait locus (QTL) for stem strength located within the chromosomal region of 28,400,000 to 28,600,000 on chromosome 3. The SCM3 gene is situated precisely within this interval, thereby confirming that the SCM3 gene is the key gene responsible for the lodging resistance trait observed in Yangchannuo 1. Development and utilization of KASP markers for the SCM3 , BADH2 , and Wx mp genes in breeding programs Resequencing analysis of two rice varieties, Yangchan 93033 and Yangchannuo 1, identified two variant sites in the SCM3 gene when compared to the reference genome NBP. One of these variants is a previously documented functional variant (TGTG/-) located at base pair 28428731 on chromosome 3 of the rice reference genome MSU7.0 (Fig. S1 ). The second variant is a single nucleotide polymorphism (vg0328430214, G/T) situated 1206 bp downstream of the ATG start codon at base pair 28430214 on the same chromosome (Fig. 4 A). An inquiry into the rice variation database indicates that vg0328430214 is found in the 3' UTR region of the SCM3 gene, occurring in only 0.7% of 4726 rice germplasm resources, 0.5% of 2759 indica rice germplasm resources, and 1.1% of 1512 japonica rice germplasm resources ( https://ricevarmap.ncpgr.cn/vars_info/ ). These findings suggest that the vg0328430214 variant in the 3' UTR of the SCM3 gene is highly specific to the natural rice population in Yangchan 93033 and Yangchannuo 1. To improve selection efficiency in breeding for lodging resistance, this specific variant can be developed into a KASP molecular marker (Table S1 ), providing a valuable tool for selecting the SCM3 gene during the enhancement of lodging resistance traits in rice. Genome resequencing analysis identified a 7-bp deletion in the second exon of the BADH2 gene (chr08:20380278) and a single nucleotide substitution (G to A) in the fourth exon of the Wx gene (chr06:1767613) in Wuxiangjing 5245 (Fig. 4 B and Fig. 4 C). These findings suggest that Wuxiangjing 5245 possesses the advantageous fragrant allele badh2_E2 and the beneficial taste allele Wx mp when compared to Yangchan 93033. Utilizing the functional variant sites of three genes, SCM3 (vg0328430214, G/T), BADH2 (vg0820380278, CGGGCGC/-------), and Wx mp (vg0601767613, G/A)—three KASP molecular markers were developed (Fig. 4 A-C). These KASP markers facilitate the rapid and efficient identification of the SCM3 , BADH2 , and Wx mp genotypes in individual plants from the Yangchan 93033/Wuxiangjing 5245 F2 population (Fig. 4 D-F). Schematic diagrams of the gene structures and locations of variation sites for SCM3 (A), BADH2 (B), and Wx (C). Genotyping results of KASP markers for SCM3 (D), BADH2 (E), and Wx mp (F). Marker-assisted selection (MAS) was employed to expedite the development of novel rice lines characterized by high yield, enhanced quality, and resistance to lodging To create new rice varieties characterized by high yield, enhanced quality, and resistance to lodging, we utilized the elite parental lines Yangchan 93033 and Wuxiangjing 5245 to produce F 1 hybrids through controlled crosses. Following self-pollination and seed harvesting, we established an F 2 segregating population. We cultivated 1,000 F 2 individuals and performed genotyping using three molecular markers: SCM3_k_28430214, BADH2_k_20380278, and Wx_mp_k_1767613, to identify the genotypes associated with the SCM3 , BADH2 , and Wx mp genes, respectively. Upon reaching maturity, we evaluated the agronomic traits of the rice plants and selected individuals that possessed favorable alleles of SCM3 , BADH2 , and Wx mp , exhibiting superior overall traits for seed collection. Ultimately, 156 individuals were selected and planted as F 3 head rows, with each head row comprising three rows of 20 seedlings each. The agronomic characteristics of 156 head rows were assessed, resulting in the identification and tagging of 500 plants exhibiting outstanding overall agronomic traits. Leaf samples were gathered for DNA extraction, and the genotypes of SCM3 , BADH2 , and Wx-mp were analyzed. In conclusion, 58 plants possessing the advantageous alleles of SCM3 , BADH2 , and Wx mp , along with exceptional comprehensive traits, were selected and progressed to the F 4 generation for additional evaluation. A total of 58 F 4 head rows were assessed for agronomic characteristics and genotyped for the SCM3 , BADH2 , and Wx mp loci. From this evaluation, 202 plants exhibiting superior overall traits and possessing the advantageous alleles of SCM3 , BADH2 , and Wx mp were selected for advancement to the F 5 generation. To minimize genotyping expenses, only those individuals with heterozygous genotypes from the preceding generation were subjected to genotyping. The 202 F 5 head rows were subsequently evaluated for agronomic traits and genotyped for SCM3 , BADH2 , and Wx mp , leading to the identification of 297 homozygous plants that carried the favorable alleles of SCM3 , BADH2 , and Wx mp , which were then advanced to the F 6 generation. In the 297 F 6 head rows, agronomic characteristics including heading date, plant height, and panicle type were assessed, leading to the selection of lines exhibiting high uniformity and the absence of segregation. A total of 44 stable lines were identified for the evaluation of lodging resistance, aimed at investigating the impact of SCM3 genotype selection on enhancing lodging resistance in rice (Table S4). The breaking resistance of the 44 F 6 lines was found to be significantly greater than that of Wuxiangjing 5245 (Fig. S5). A comparative analysis was conducted on various traits, including the number of productive panicles (A), stem breaking resistance (B), lodging index (C), length of the second basal internode (D), diameter of the second basal internode (E), and wall thickness of the second basal internode (F), among 44 F 6 progeny lines and their parental varieties, Yangchan 93033 and Wuxiangjing 5245. Welch's Two Sample t -test was employed to assess the significance of the observed differences. Assessment of agronomic characteristics in stable lines A comparative analysis was conducted on the number of productive panicles (A), total grains per panicle (B), 1000-grain weight (C), and yield per plant (D) across eight stable elite lines. The least significant difference at the 0.01 level (LSD_0.01) was employed for multiple comparison analysis. We identified eight stable lines exhibiting outstanding overall characteristics and superior lodging resistance, based on their lodging resistance performance and other agronomic traits, for a comprehensive evaluation of agronomic traits and an analysis of yield and quality. The number of productive panicles in the lines 24HD134 and 24HD362 was significantly greater than that observed in Wuxiangjing 5245 and Yangchan 93033 (Fig. 6 A). In contrast, the remaining lines exhibited a notable decrease in productive panicle count compared to the two parental varieties (Fig. 6 A). All eight stable lines demonstrated a significantly higher number of grains per panicle than Wuxiangjing 5245, with three lines exhibiting values comparable to Yangchan 93033, showing no significant differences (Fig. 6 B). However, the 1000-grain weight for all eight stable lines was found to be lower than that of Yangchan 93033 (Fig. 6 C). Specifically, the 1000-grain weight of 24HD127 was significantly less than that of both Wuxiangjing 5245 and Yangchan 93033, while the other seven lines did not show significant differences when compared to Wuxiangjing 5245 (Fig. 6 C). Among the eight lines studied, the yield per plant for 24HD134, 24HD142, 24HD365, and 24HD448 was significantly greater than that of Wuxiangjing 5245, although it did not surpass that of Yangchan 93033 (Fig. 6 D). In conclusion, the varieties 24HD134, 24HD142, 24HD365, and 24HD448 showed a notable enhancement in both the number of grains per panicle and the yield per plant when compared to Wuxiangjing 5245, indicating their considerable yield potential. A comparative analysis was conducted on the brown rice yield (A), milled rice yield (B), sensory evaluation of cooked rice (C), and amylose content in rice flour (D) across eight stable elite lines. The Least Significant Difference (LSD) at the 0.01 level was employed for multiple comparison assessments. The brown rice yield of 24HD127 was significantly lower than that of Wuxiangjing 5245, whereas the remaining seven lines did not show any significant differences when compared to Wuxiangjing 5245 (Fig. 7 A). Among the eight stable lines, 24HD362 demonstrated the highest milled rice yield, which was significantly greater than that of Wuxiangjing 5245 (Fig. 7 B). The cooked rice taste value for 24HD134, 24HD146, and 24HD362 was significantly superior to that of Yangchan 93033 (Fig. 7 C). Additionally, the amylose content in 24HD134, 24HD146, 24HD362, 24HD365, and 24HD448 was lower than that found in Yangchan 93033 (Fig. 7 D). A comparative evaluation of stem breaking resistance (A), lodging index (B), diameter of the second basal internode (C), and wall thickness of the second basal internode (D) was conducted among eight stable elite lines. The least significant difference at the 0.01 level (LSD_0.01) was employed for the multiple comparison analysis. The breaking strength of the stems (BS) in all eight stable lines was significantly greater than that of Wuxiangjing 5245, yet it did not surpass that of Yangchan 93033 (Fig. 8 A). The lodging index for lines 24HD146, 24HD362, and 24HD448 was significantly higher than that of Yangchan 93033, whereas the lodging index for line 24HD127 was significantly lower than that of Yangchan 93033 (Fig. 8 B). The remaining four lines exhibited no significant differences in lodging index when compared to Yangchan 93033(Fig. 8 B). The diameter of the second basal internode in the lines 24HD142, 24HD146, 24HD362, and 24HD448 was significantly greater than that observed in Wuxiangjing 5245 (Fig. 8 C). Additionally, the wall thickness of the second basal internode in the lines 24HD112, 24HD142, 24HD146, 24HD362, 24HD365, and 24HD448 was notably thicker compared to Wuxiangjing 5245 (Fig. 8 D). The experimental findings indicate that the 24HD134 line exhibits outstanding overall performance in terms of yield, quality, and resistance to lodging. This positions it as a strong candidate for subsequent production trials as a new line characterized by high yield, superior quality, and enhanced lodging resistance. Discussion As rice variety yields continue to improve, the susceptibility to lodging in high-yielding varieties under extreme climatic conditions has intensified. To expedite the breeding of high-yielding and stable varieties, we systematically assessed lodging resistance traits in 78 japonica rice germplasm resources. Our screening identified 14 rice materials with the SCM3 9311 genotype, which exhibited notable lodging resistance characteristics. These materials show considerable breeding potential and can be utilized as important parental resources for the development of lodging-resistant varieties. A comprehensive investigation and analysis of the agronomic traits of 14 rice varieties with the SCM3 9311 genotype revealed that Yangchan 93033 and Yangchannuo 1 possess exceptional overall characteristics. These two varieties are characterized by sturdy stems, strong resistance to bending, a high number of grains per panicle, and elevated single-plant yields, indicating considerable yield potential. Consequently, Yangchan 93033 and Yangchannuo 1 represent valuable germplasm resources for lodging resistance, offering crucial genetic materials for the development of high-yield and stable rice varieties. Through Bulked Segregant Analysis (BSA), we have confirmed that the primary gene responsible for stem diameter in Yangchannuo 1 is SCM3 . A comparative analysis of the SCM3 gene sequences from Yangchannuo 1 and Yangchan 93033 identified a unique single nucleotide polymorphism (SNP) site (vg0328430214, G/T) present in both varieties. This SNP is significantly linked to the robust stem characteristic and can be further developed into a Kompetitive Allele-Specific PCR (KASP) molecular marker. The establishment of this marker will enable precise screening for lodging resistance and facilitate molecular marker-assisted breeding, thereby providing essential technical support and genetic resources for the development of high-yield and stable rice varieties. The notable enlargement of the cross-sectional area of vascular bundles in rice stems significantly enhances the transport efficiency of non-structural carbohydrates, which in turn promotes dry matter accumulation and ultimately boosts yield (Li et al., 2022 ). Specifically, in the case of Yangchan 93033, the cross-sectional area of vascular bundles in the second internode at the base is considerably larger compared to Wuxiangjing 5245. This structural advantage provides Yangchan 93033 with an enhanced capacity for nutrient transport, thereby facilitating dry matter accumulation and leading to a substantial increase in yield. A progeny population was developed from the cross between Yangchan 93033 and Wuxiangjing 5245, and molecular marker-assisted selection was utilized to evaluate 44 lines from the F 6 generation. These lines demonstrated a notable enhancement in stem breaking resistance and stem diameter when compared to Wuxiangjing 5245, indicating that the SCM3 molecular marker can effectively and swiftly improve lodging resistance in breeding materials. Nonetheless, the breaking resistance of these lines was still significantly inferior to that of Yangchan 93033, which may be attributed to variations in genetic background. Consequently, for future breeding efforts aimed at improving lodging resistance, Yangchan 93033 should be considered as a recurrent parent for backcrossing with high-yield and high-quality materials to fully leverage its advantageous lodging resistance characteristics. Subsequent evaluation of agronomic traits led to the identification of eight stable lines from the aforementioned population, which were thoroughly assessed for yield and quality. Notably, line 24HD134 demonstrated exceptional performance in terms of yield, flavor quality, and resistance to lodging, resulting in a synergistic enhancement of these traits. This line represents a significant resource for the development of rice varieties that are high-yielding, high-quality, and resistant to lodging. In the future, 24HD134 can be used as a parent line to combine Gn1a/OsCKX2 , STRONG2 and SCM2 through molecular marker-assisted selection, thereby cultivating high-yielding and high-quality rice varieties with improved resistance to lodging. While molecular marker-assisted selection facilitates the swift and effective identification of desired traits, the stabilization of other agronomic characteristics within breeding populations still necessitates several generations of self-fertilization. To expedite the breeding process, future initiatives may explore the use of pollen microspore culture or haploid induction methods to generate doubled haploid lines, which would substantially improve breeding efficiency (Chen et al., 2024 ). In August 2024, Yangzhou experienced an average maximum temperature of 36°C, resulting in high-temperature stress during the heading phase around August 18. This stress significantly reduced both the seed setting rate and grain quality in the affected rice lines. The eight stable lines analyzed in this study demonstrated notable variations in their growth duration. To thoroughly assess varietal adaptability, lines that headed around August 18 are recommended for cultivation in northern Jiangsu Province, while those that headed around September 6 are suitable for southern Jiangsu Province. A systematic evaluation of their yield, quality, and lodging resistance will aid in the identification of superior rice lines that are well-suited to different ecological regions. Conclusions This study established a molecular marker based on the functional divergence sites of the SCM3 gene to evaluate lodging resistance in 78 rice germplasm resources. Fourteen accessions with the SCM3⁹³¹¹ genotype showed exceptional lodging resistance, particularly Yangchan 93033 and Yangchannuo 1, which exhibited strong stems and high grain yield. A KASP marker, SCM3_k_28430214, was confirmed as an effective tool for molecular marker-assisted selection. A hybrid population was created by crossing Yangchan 93033 with Wuxiangjing 5245, leading to the development of a new breeding line, 24HD134, which integrates favorable alleles and demonstrates high yield potential, superior grain quality, and improved lodging resistance. Methods Experimental Materials and Cultivation The study utilized 77 rice varieties that have either recently been approved or are currently undergoing trials in Jiangsu Province, along with breeding materials such as Yangchan 93033 and Wuxiangjing 5245. These materials were cultivated at the experimental facility located in Shatou Town, Guangling District, Yangzhou City. All rice varieties were transplanted as individual seedlings, maintaining a plant spacing of 12 cm and a row spacing of 28 cm. Nitrogen fertilizer was applied at a rate of 258.75 kg/ha of pure nitrogen, adhering to a fertilizer application ratio of 4:3:3 for basal, tiller, and panicle fertilizers. Water and fertilizer management, along with pest and disease control, were implemented in accordance with the standards for high-yield rice cultivation. Development of Functional Markers A 4-bp variation site (ID: vg0328428721, C/CTGTG) located in the promoter region of the SCM3 gene ( LOC_Os03g49880 ) was identified from publicly accessible data on the website http://ricevarmap.ncpgr.cn/ . Utilizing the flanking sequence information of the vg0328428721 variation site, a pair of primers was designed with Primer Premier 5.0 software. The development and design of the KASP (Kompetitive Allele-Specific PCR) marker were outsourced to Jingtai Biotechnology Co., Ltd., employing the sequences adjacent to the gene variation site. Molecular marker detection was conducted using standard Polymerase Chain Reaction (PCR) amplification techniques in the laboratory. The reaction mixture, totaling 10 µL, included 1 µL of template DNA (approximately 15 ng µL –1 ), 0.4 µL of each forward and reverse primer (10 µmol µL –1 ), 5 µL of 2×NG PCR MasterMix (Shanghai Huiling Biotechnology Co., Ltd., NG001M), and 3.2 µL of sterile double-distilled water. The amplification process was performed in a PCR machine under the following conditions: (1) initial denaturation at 95°C for 5 minutes; (2) 35 cycles consisting of denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds; and (3) a final extension at 72°C for 10 minutes. The resulting PCR products were analyzed by electrophoresis on an 8% polyacrylamide gel. PCR amplification of the KASP marker was conducted utilizing the PARMS mix from Wuhan Jingtai Biotechnology. The reaction system, totaling 10 µL, comprised approximately 50 ng of template DNA, 0.15 µL each of two allele-specific primers (10 µmol L –1 ), 5 µL of 2×PARMS master mix (Wuhan Jingtai Biotechnology), and sterile double-distilled water to achieve the final volume. The amplification process was executed in a PCR instrument under the following conditions: (1) initial denaturation at 94°C for 15 minutes; (2) 10 cycles of denaturation at 94°C for 20 seconds, followed by annealing at a temperature decreasing from 65°C to 57°C (decreasing by 0.8°C per cycle) for 60 seconds; and (3) 32 cycles of denaturation at 94°C for 20 seconds and annealing at 57°C for 60 seconds. Upon completion of the PCR, fluorescence signals were detected using a TECAN Infinite M1000 microplate reader. These signals were subsequently analyzed and transformed into clear and intuitive genotyping plots via the online software snpdecoder ( http://www.snpway.com/snpdecoder/ ). The genotyping results were generated based on the observed color differences. Evaluation of Stem Robustness in Rice Cultivars The heading date of the test materials was meticulously documented. At 25 days post-heading, three plants from each material were chosen to assess plant height (PH) and the number of panicles per plant (PN). The main stems, along with the fibrous roots, were severed and promptly placed in water to avoid dehydration before being transported to the laboratory for the measurement of stem traits. The collected stems were positioned on the compression force gauge holder of a plant stem strength tester (Zhejiang Top Instrument Co., Ltd., model: YYD-1A), with the distance between the two support points adjusted to 9 cm. The handle of the force gauge was pressed downward until the stem fractured, and the maximum pressure recorded was designated as the bending stress (BS). A ruler and balance were utilized to measure the length from the fractured section of the basal stem to the apex of the panicle (Stem Length, SL) and to determine the fresh weight (FW). The lodging index (LI) was calculated using the formula: LI = SL × FW / (BS × 9) / 4. The length of the second internode from the plant root (IL2) was measured with a ruler. The outer diameters of the second internode, both short-axis and long-axis (excluding the leaf sheath), were measured using a vernier caliper, and the average was recorded as the culm outer diameter at the cross-section of the second internode (CD2). Likewise, the thicknesses of the cross-section of the second internode (excluding the leaf sheath) were measured for both short-axis and long-axis using a vernier caliper, with the average value documented as the culm thickness at the cross-section of the second internode (CT2). Assessment of Rice Crop Production Upon reaching full maturity, ten main stem panicles were randomly selected from healthy plants located in the central section of each row. These harvested panicles were placed in mesh bags for drying. Following the drying process, the grains were threshed, and both unfilled and filled grains were counted separately. The total grain count per panicle (NGP) was determined by adding the numbers of unfilled and filled grains. The seed setting rate (SP) was calculated as the ratio of filled grains to the total grain count per panicle. The filled grains from each main stem panicle were preserved and subsequently analyzed using a grain analyzer to assess characteristics such as grain length, grain width, and the weight of 1000 grains. Furthermore, ten healthy plants from each line were randomly chosen, harvested, and placed in mesh bags for the drying process. The dried plants were then threshed using a small thresher, and the grains from each plant were weighed individually. The average grain weight per plant was computed and documented as the single plant yield (YP) for each line. Imaging Cross-Sections of the Rice Stem Base At 30 days after heading, representative main tillers were collected and the leaf sheaths were carefully removed. The basal second internode was excised and immediately fixed in FAA solution (70% ethanol 90 mL, glacial acetic acid 5 mL, formaldehyde 5 mL) for 24 h to preserve cellular integrity. Samples were then softened in an appropriate maceration solution until fully pliable, followed by dehydration and paraffin embedding. Serial sections were cut and stained in safranin for 1–2 h. Excess stain was removed by rinsing in tap water, and sections were briefly decolorized (3–8 s each) through a graded ethanol series (50%, 70%, 80%). Thereafter, sections were counterstained in fast green for 30–60 s, dehydrated through three changes of absolute ethanol, and cleared in fresh xylene for 5 min. Finally, slides were mounted with neutral gum for long-term preservation. Observations and photomicrographs were obtained under a Leica S8 APO stereomicroscope, and morphological measurements of vascular bundle number, area, and perimeter were carried out on the acquired images. Assessment of Rice Processing, Visual Characteristics, and Flavor Quality Determination of Brown Rice Yield: Following the harvesting process, the rice undergoes threshing and sun-drying before being stored in a controlled indoor environment for a duration of three months. A sample weighing 150 grams is extracted from each batch. The brown rice is produced by milling the sample with a rice husker, and the weight of the resulting brown rice is recorded to facilitate the calculation of the brown rice yield. This procedure is replicated three times, ensuring that the discrepancy between the first and third measurements remains within 2%. The final result is derived from the average of the three measurements. The brown rice yield is calculated using the formula: Brown Rice Yield = (Weight of Brown Rice (g) / 150g) × 100% Determination of Milled Rice Rate: The brown rice produced in the preceding step undergoes additional milling with a rice polisher, and the weight of the resulting milled rice is documented. This procedure is conducted three times, ensuring that the discrepancy between the first and third measurements does not exceed 1%. The final result is derived from the average of these three measurements. The milled rice rate is computed using the following formula: Milled Rice Rate = (Weight of Milled Rice (g) / 150g) × 100% Determination of Transparency, Chalkiness, and Chalky Grain Rate: A random sample of 100 intact milled rice grains is uniformly distributed on the scanning plate of the rice appearance quality analyzer. The Wanshen SC-E Rice Appearance Quality Analyzer software is utilized to assess transparency, chalkiness, and the chalky grain rate. Each measurement is conducted in triplicate, ensuring that the error for transparency remains within 2%, for chalkiness within 10%, and for the chalky grain rate within 5%. The final result is derived from the average of the three measurements. Taste Quality Assessment: A 30g sample of intact milled rice is placed in a stainless steel container, rinsed, and subsequently combined with water at a ratio of 1:1.3. The container is sealed using a rubber ring and filter paper, and the mixture is soaked for 30 minutes. Following this, the rice is steamed for 30 minutes, allowed to rest for 10 minutes, and then cooled for 20 minutes utilizing a forced air cooling system. After resting at room temperature for 90 minutes, an 8g sample of the cooked rice is extracted, and taste quality along with related parameters is evaluated using a rice taste analyzer (STA1A) manufactured by Satake Corporation. This procedure is repeated three times, and the average value is recorded as the final result. Assessment of Amylose Levels in Rice Determination of Amylose Content: Weigh 0.05 g of rice flour that has been sieved through a 100-mesh screen and transfer it into a 50 mL digestion tube. Add 0.5 mL of 95% ethanol and 4.5 mL of 1 mol/L NaOH to the tube. Heat the mixture in a boiling water bath for 10 minutes, then allow it to cool to room temperature. Finally, dilute the solution with distilled water to achieve a final volume of 50 mL, thereby preparing the sample solution. Transfer 2.5 mL of the sample solution into a 50 mL digestion tube. Acidify the mixture by adding 0.5 mL of 1 mol/L acetic acid solution, followed by the addition of 0.75 mL of iodine solution. Mix thoroughly and dilute with distilled water to achieve a final volume of 50 mL. Let the solution sit for 20 minutes. Adjust the spectrophotometer to a wavelength of 620 nm and calibrate it using a blank solution. Subsequently, measure the absorbance of the sample. Repeat this procedure with a standard sample that has a known amylose content to obtain its absorbance and create a standard curve. The amylose content of the sample is then determined using this standard curve. Data Analysis Data processing and statistical analyses were conducted using Microsoft Excel 2013. The t .test function from the tidyr R package was employed to assess significant differences between groups. The analysis of variance (ANOVA) for multiple comparisons was conducted using the AOV analysis module of the QTL IciMapping software, which can be accessed at https://isbreeding.caas.cn/rj/index.htm . Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare that they have no conflict of interest. Funding This work was supported by the Project funded by National Key Research and Development Program of China (2022YFD1200104), China Postdoctoral Science Foundation (2021M702767), and Revitalization of Seed Industry in Jiangsu Province: JBGS Project（JBGS［2021］036）. Authors ’ contributions HJL, ZJY, LGH and ZY conceived and designed the experiments. HJL, ZY, ZZX, MP, ZX, ZNB and ZJY performed the experiments and analyzed the data. HJL, ZY and ZJY was responsible for material plant and field management. HJL wrote the manuscript. WHY, ZY, LGH and ZHC revised the manuscript. All authors read and approved the manuscript. References Asano K, Takashi T, Miura K, Qian Q, Kitano H, Matsuoka M, Ashikari M (2007) Genetic and molecular analysis of utility of sd1 alleles in rice breeding. Breeding Science , 57 (1), 53–58. https://doi.org/10.1270/jsbbs.57.53 Chen, J., Li, S., Zhou, L., Zha, W., Xu, H., & Liu, K. (2024). 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S2.jpg Fig. S2 Evaluation of agronomic characteristics in 14 japonica rice varieties harboring the SCM3 9311 genotype A comparative analysis was conducted on various agronomic traits of 14 japonica rice materials possessing the SCM3 9311 genotype. The traits examined included the number of grains per panicle (A), 1000-grain weight (B), grain length (C), grain width (D), plant height (E), and yield per plant (F). For the multiple comparison analysis, a significance level of LSD_0.01 was employed. S3.jpg Fig. S3 Comparison of agronomic characteristics between two japonica rice varieties: Yangchan 93033 and Wuxiangjing 5245 A comparative analysis was conducted on various parameters between two japonica rice varieties, Yangchan 93033 and Wuxiangjing 5245. The parameters examined included the diameter of the second basal internode (A), wall thickness of the second basal internode (B), stem breaking resistance (C), length of the second basal internode (D), plant height (E), lodging index (F), number of grains per panicle (G), 1000-grain weight (H), grain length (I), grain width (J), number of productive panicles (K), and yield per plant (L). Welch's Two Sample t -test was employed to assess the significance of the differences observed. S4.jpg Fig. S4 Bulk segregant analysis (BSA) of traits associated with lodging resistance in Yangchannuo 1 S5.jpg Fig. S5 Comparison of agronomic characteristics among 44 F 6 progeny lines and their parental lines A comparative analysis was conducted on various traits, including the number of productive panicles (A), stem breaking resistance (B), lodging index (C), length of the second basal internode (D), diameter of the second basal internode (E), and wall thickness of the second basal internode (F), among 44 F 6 progeny lines and their parental varieties, Yangchan 93033 and Wuxiangjing 5245. Welch's Two Sample t -test was employed to assess the significance of the observed differences. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6845239","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":471657284,"identity":"18046ada-12b1-4803-a503-a1feabd441c5","order_by":0,"name":"Jinlong Hu","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Jinlong","middleName":"","lastName":"Hu","suffix":""},{"id":471657285,"identity":"27cb4898-471a-4e83-aefd-cc57a161427c","order_by":1,"name":"Zexu Zhou","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zexu","middleName":"","lastName":"Zhou","suffix":""},{"id":471657286,"identity":"f940cc6e-a756-4d70-bb11-2d6b51be8bc8","order_by":2,"name":"Yu Zhang","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":471657287,"identity":"93b2ae76-dff0-46a2-9f77-182978247548","order_by":3,"name":"Pan Mao","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Pan","middleName":"","lastName":"Mao","suffix":""},{"id":471657288,"identity":"7e77982c-3036-4d0e-869d-a5c3903e24fd","order_by":4,"name":"Xin Zhao","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhao","suffix":""},{"id":471657289,"identity":"0f05e30c-bf5e-41bf-b938-1ff3135106f4","order_by":5,"name":"Nianbing Zhou","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Nianbing","middleName":"","lastName":"Zhou","suffix":""},{"id":471657290,"identity":"075eb544-0294-4336-a32b-cdc003cfad7f","order_by":6,"name":"Qiangqiang Xiong","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Qiangqiang","middleName":"","lastName":"Xiong","suffix":""},{"id":471657291,"identity":"033c287c-bd77-4f25-8d1b-76440eee598f","order_by":7,"name":"Yong Zhou","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Zhou","suffix":""},{"id":471657292,"identity":"ced7fc95-fbd2-40e7-9df5-053d8c910828","order_by":8,"name":"Haiyan Wei","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Wei","suffix":""},{"id":471657293,"identity":"704d52fc-5d7c-4109-9a45-f61e4535cc07","order_by":9,"name":"Hongcheng Zhang","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Hongcheng","middleName":"","lastName":"Zhang","suffix":""},{"id":471657294,"identity":"fd88e843-341a-4ed9-87b1-025ec0ab0a13","order_by":10,"name":"Guohua Liang","email":"","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":false,"prefix":"","firstName":"Guohua","middleName":"","lastName":"Liang","suffix":""},{"id":471657296,"identity":"da2c47f4-6a20-4325-a990-b128b9a0331e","order_by":11,"name":"Jinyan Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYDACCQYGgwQo+wBDhYScPIlazlgYGzYQoQUBGNsqEoEa8QP52T0GBQ93HJY3lz788HDhPIkExgbmh49u4NHCOOeMgUHimcOGO/vSDA7P3CaRx87AZmycg0cLs0QOUEvbYcYNZxgMDvNukyhmbOBhk8anhQ2qxX7DGfYPh3nnSCQ2HCCghQeqJXHDGR6gLQ1EaJGQSCsAaklPBmopOMxzTMLYsJmAX+RnJG8z/NlmbQt02ObPPDV1cvLszQ8f49MC8o4BA0MzEp8Zv3KwkgcMDHWElY2CUTAKRsHIBQDKv0sEy2/mUAAAAABJRU5ErkJggg==","orcid":"","institution":"Agricultural College of Yangzhou University","correspondingAuthor":true,"prefix":"","firstName":"Jinyan","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2025-06-08 03:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6845239/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6845239/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":84799451,"identity":"8c2482c3-b342-44b3-8158-9ba5ee450587","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":166668,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of lodging resistance characteristics between two scm3 genotypes in 78 japonica rice varieties\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis was conducted on various parameters, including the diameter of the second basal internode (A), wall thickness of the second basal internode (B), stem breaking resistance (C), length of the second basal internode (D), plant height (E), and lodging index (F) between the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e genotypes across 78 japonica rice materials. Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the observed differences.\u003c/p\u003e","description":"","filename":"Picture1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/92bdde6e84f7192f39c57b6d.jpg"},{"id":84799450,"identity":"36719bd6-a7e1-4c2a-8f6a-f5aef3431b4b","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":182848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of yield-associated traits between two \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSCM3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genotypes in 78 japonica rice varieties\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis was conducted on various traits, including the number of grains per panicle (A), 1000-grain weight (B), grain length (C), grain width (D), plant height (E), and yield per plant (F), between the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e genotypes across 78 japonica rice materials. Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the observed differences.\u003c/p\u003e","description":"","filename":"Picture2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/de8a4ab34701dd8b52eb039d.jpg"},{"id":84799455,"identity":"9ba2d553-1825-430c-a02a-21e92b424be6","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":295743,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eVascular bundle architecture of two japonica rice varieties, Yangchan 93033 and Wuxiangjing 5245\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/d4a3bfc2cbc1bc275c004fc8.jpg"},{"id":84799472,"identity":"f997a199-d9ab-423b-99db-3eb5182b74f0","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":152877,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene architecture and development of KASP markers for functional regions of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSCM3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBADH2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eWx\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003emp\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e genes\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic diagrams of the gene structures and locations of variation sites for \u003cem\u003eSCM3\u003c/em\u003e (A), \u003cem\u003eBADH2\u003c/em\u003e (B), and \u003cem\u003eWx\u003c/em\u003e (C). Genotyping results of KASP markers for \u003cem\u003eSCM3\u003c/em\u003e (D), \u003cem\u003eBADH2\u003c/em\u003e (E), and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e (F).\u003c/p\u003e","description":"","filename":"Picture4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/7a5367282b4cb96cded89936.jpg"},{"id":84799459,"identity":"be64d7c5-52cf-4b11-abbe-f0a01194d122","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":98826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlowchart for the Breeding of MAS to enhance yield, quality, and lodging resistance in the progeny population of Yangchan 93033/Wuxiangjing 5245\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/6852b8e85ddce6b945a3e6ef.jpg"},{"id":84799683,"identity":"02197e67-8f3b-4180-9bb6-40d1e4e4cd0e","added_by":"auto","created_at":"2025-06-17 13:04:43","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":112501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of yield-associated characteristics among eight stable elite lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis was conducted on the number of productive panicles (A), total grains per panicle (B), 1000-grain weight (C), and yield per plant (D) across eight stable elite lines. The least significant difference at the 0.01 level (LSD_0.01) was employed for multiple comparison analysis.\u003c/p\u003e","description":"","filename":"Picture6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/b72c1f795c6d377d7c5513dd.jpg"},{"id":84800499,"identity":"1b716424-5d13-47b4-a454-94688c9eb8f5","added_by":"auto","created_at":"2025-06-17 13:12:44","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":111484,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of quality characteristics among eight elite lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis was conducted on the brown rice yield (A), milled rice yield (B), sensory evaluation of cooked rice (C), and amylose content in rice flour (D) across eight stable elite lines. The Least Significant Difference (LSD) at the 0.01 level was employed for multiple comparison assessments.\u003c/p\u003e","description":"","filename":"Picture7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/6761615d50c4ad90c63f1d95.jpg"},{"id":84799470,"identity":"23e586df-999e-41cd-ab3d-d1d9ed0f0846","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":116364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of lodging resistance characteristics across eight elite varieties\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative evaluation of stem breaking resistance (A), lodging index (B), diameter of the second basal internode (C), and wall thickness of the second basal internode (D) was conducted among eight stable elite lines. The least significant difference at the 0.01 level (LSD_0.01) was employed for the multiple comparison analysis.\u003c/p\u003e","description":"","filename":"Picture8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/cccf94b33790a19e6290883b.jpg"},{"id":85726704,"identity":"6d964b5c-faf9-4741-bf26-ae342ff01f43","added_by":"auto","created_at":"2025-07-01 06:47:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2243898,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/9f815ec2-79c4-4dc1-9fc4-a89c3591b4eb.pdf"},{"id":84800496,"identity":"30311a5f-b279-4b8e-9110-90d161786c86","added_by":"auto","created_at":"2025-06-17 13:12:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":50293,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2025.6.8.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/963d02d3977d7af9aa04639f.xlsx"},{"id":84800495,"identity":"b7491817-bfc4-4f01-906b-e4639d81b0ed","added_by":"auto","created_at":"2025-06-17 13:12:43","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":732691,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryfulluncroppedGels.docx","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/2c04df5878000f3066453797.docx"},{"id":84800494,"identity":"cdeee084-0041-4193-9046-788cbbbccb53","added_by":"auto","created_at":"2025-06-17 13:12:43","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":63961,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S1 Gene architecture of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSCM3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and the advancement of functional markers\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. The gene structure of \u003cem\u003eSCM3\u003c/em\u003e and the positions of functional variation sites. B. Polyacrylamide gel electrophoresis analysis of SCM3_indel_2.\u003c/p\u003e","description":"","filename":"S1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/2f6f30d4133aa46f55268de9.jpg"},{"id":84799682,"identity":"f96eabc9-41fc-43d9-8969-d2d6793abd53","added_by":"auto","created_at":"2025-06-17 13:04:43","extension":"jpg","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":278879,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S2 Evaluation of agronomic characteristics in 14 japonica rice varieties harboring the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSCM3\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e9311\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e genotype\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis was conducted on various agronomic traits of 14 japonica rice materials possessing the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e genotype. The traits examined included the number of grains per panicle (A), 1000-grain weight (B), grain length (C), grain width (D), plant height (E), and yield per plant (F). For the multiple comparison analysis, a significance level of LSD_0.01 was employed.\u003c/p\u003e","description":"","filename":"S2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/e8ee9ee0541c5a6cae75ec5c.jpg"},{"id":84799466,"identity":"6b3b8bbd-1319-4a6e-9f58-5f811f030409","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":135242,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S3 Comparison of agronomic characteristics between two japonica rice varieties: Yangchan 93033 and Wuxiangjing 5245\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis was conducted on various parameters between two japonica rice varieties, Yangchan 93033 and Wuxiangjing 5245. The parameters examined included the diameter of the second basal internode (A), wall thickness of the second basal internode (B), stem breaking resistance (C), length of the second basal internode (D), plant height (E), lodging index (F), number of grains per panicle (G), 1000-grain weight (H), grain length (I), grain width (J), number of productive panicles (K), and yield per plant (L). Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the differences observed.\u003c/p\u003e","description":"","filename":"S3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/b5beb25fe36ae7df5ba9c18f.jpg"},{"id":84799467,"identity":"24bebcc7-b732-4f23-ada6-0445bb751c69","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":460374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S4 Bulk segregant analysis (BSA) of traits associated with lodging resistance in Yangchannuo 1\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"S4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/f1e41211621ae0e4ef097d0e.jpg"},{"id":84799456,"identity":"1f932772-0d89-4b3b-9a87-5531e8a61c01","added_by":"auto","created_at":"2025-06-17 12:56:43","extension":"jpg","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":121765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. S5 Comparison of agronomic characteristics among 44 F\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e progeny lines and their parental lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparative analysis was conducted on various traits, including the number of productive panicles (A), stem breaking resistance (B), lodging index (C), length of the second basal internode (D), diameter of the second basal internode (E), and wall thickness of the second basal internode (F), among 44 F\u003csub\u003e6\u003c/sub\u003e progeny lines and their parental varieties, Yangchan 93033 and Wuxiangjing 5245. Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the observed differences.\u003c/p\u003e","description":"","filename":"S5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6845239/v1/2c60a7067b8b60d5ca69d63e.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular Marker-Assisted Pyramiding of SCM3, Wx, and BADH2 Genes for the Development of High-Yield, Superior-Quality, and Lodging-Resistant Rice Varieties","fulltext":[{"header":"Background","content":"\u003cp\u003eRice is a crucial staple crop in China, with its high and stable yield being essential for national food security. Lodging, which can occur during the mid to late growth stages, is influenced by environmental conditions, cultivation practices, and the genetic traits of the rice varieties. Mild lodging causes the plants to lean, resulting in reduced light penetration within the canopy and a decline in photosynthetic efficiency. In severe cases, broken stems hinder nutrient transport and water absorption, hastening the aging of the rice plants. As a result, lodging leads to inadequate grain filling and a substantial decrease in yield (Guo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, when rice panicles are positioned close to the ground, it can result in grain mold or pre-harvest sprouting, negatively impacting rice quality and reducing seed germination rates, while also increasing harvesting costs (Mullangie et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLodging in rice is primarily influenced by four key factors. Firstly, adverse environmental conditions such as heavy rainfall and strong winds can cause rice plants to lean or even break at the stem. Secondly, varietal traits play a significant role; tall varieties with large panicles often possess a higher center of gravity, which compromises their resistance to lodging (Merugumala et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, varieties characterized by thin and soft stems, elongated basal internodes, wide tillering angles, loose plant structures, and shallow root systems tend to have diminished lodging resistance (Mullangie et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Thirdly, cultivation management practices are critical; excessive nitrogen fertilizer application can lead to increased plant height and elongated basal internodes, while simultaneously reducing stem wall thickness and diameter, thereby weakening the lodging resistance of rice (Zhang et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). High planting densities may also result in taller plants with thinner, weaker stems, as well as decreased dry weight and mechanical strength per internode, further impairing the plant's lodging resistance. Lastly, planting methods can impact lodging; simplified cultivation techniques, such as direct seeding, may lead to uneven sowing and shallow seed placement, resulting in overcrowding and shallow root systems, which further exacerbate lodging issues (Yadav et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe development and cultivation of lodging-resistant rice varieties are recognized as economically viable strategies to mitigate the issue of rice lodging. The resistance of rice to lodging is primarily influenced by characteristics such as plant height, stem diameter, stem strength, and tillering angle (Sowadan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Mulsanti et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Guo et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mullangie et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). A negative correlation exists between plant height and lodging resistance; taller plants possess a higher center of gravity, rendering them more prone to lodging. The stem serves as the principal support structure for rice, and varieties with thicker stems typically demonstrate enhanced lodging resistance (Samadi et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mullangie et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In the 1960s, the introduction of semi-dwarf genes effectively addressed the lodging issue in rice (Asano et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Nevertheless, the biomass accumulation of semi-dwarf rice varieties is constrained by their reduced height, which limits further yield improvements. Consequently, a strategic increase in plant height can boost the biological yield of rice, while enhancing stem thickness and strength can improve lodging resistance. This strategy facilitates an increase in yield potential while simultaneously mitigating the risk of lodging.\u003c/p\u003e \u003cp\u003eTo date, several quantitative trait loci (QTLs) associated with stem thickness and strength in rice have been successfully cloned, including \u003cem\u003eSCM2\u003c/em\u003e, \u003cem\u003eSCM3\u003c/em\u003e/\u003cem\u003eOsTB1\u003c/em\u003e, and \u003cem\u003eIPA1\u003c/em\u003e, among others. \u003cem\u003eSCM2\u003c/em\u003e encodes an F-box protein predominantly expressed in the apical meristem and lateral organ primordia (Ookawa et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Enhanced expression of \u003cem\u003eSCM2\u003c/em\u003e results in increased stem diameter, improved mechanical strength, and a notable rise in the number of grains per panicle (Ookawa et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eSCM3\u003c/em\u003e encodes a transcription factor from the TCP family and functions as a negative regulator of tiller number in rice. The \u003cem\u003eSCM3\u003c/em\u003e allele found in indica varieties, such as 9311 and Chugoku 117, possesses a 4-bp insertion in the 5' UTR region, leading to elevated expression levels that contribute to greater stem thickness and strength. In contrast, japonica varieties like Nipponbare and Koshihikari lack this 4-bp insertion, resulting in lower \u003cem\u003eSCM3\u003c/em\u003e expression and thinner stems (Yano et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). By employing CRISPR/Cas9 gene editing technology to modify the promoter region of \u003cem\u003eSCM3\u003c/em\u003e, researchers have developed favorable allelic variants with increased gene expression, which in turn enhances stem thickness and the number of grains per panicle (Cui et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ideal plant architecture gene \u003cem\u003eIPA1\u003c/em\u003e encodes a Squamosa promoter-binding protein-like transcription factor, OsSPL14, which directly binds to the promoter of \u003cem\u003eSCM3\u003c/em\u003e to inhibit tiller formation (Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Gene editing of the \u003cem\u003eIPA1\u003c/em\u003e promoter has led to the creation of novel allelic variants with increased expression levels, resulting in simultaneous enhancements in panicle weight and grain number, as well as thicker stems and roots (Song et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Loss of function of Gn1a/OsCKX2 leads to the accumulation of cytokinins in the crown root tips, accelerating the development of adventitious roots, increasing the number of grains per panicle in rice plants, and exhibiting excellent lodging resistance (Tu et al., 2022; J. Zhang et al., 2024).\u003c/p\u003e \u003cp\u003eRecent research has successfully cloned the \u003cem\u003eSTRONG2\u003c/em\u003e gene, known for its high yield and resistance to lodging, through genome-wide association studies (GWAS). This gene encodes a cellulose synthase-like enzyme (CSLA) that plays a crucial role in the development of the secondary cell wall by modulating the composition of the cell wall, which in turn enhances stem thickness and strength (Zhao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Furthermore, the analysis indicated that the beneficial allele of \u003cem\u003eSTRONG2\u003c/em\u003e is predominantly found in improved indica rice varieties, whereas its occurrence is comparatively lower in enhanced japonica rice varieties (Zhao et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Consequently, the \u003cem\u003eSTRONG2\u003c/em\u003e gene presents significant potential for future genetic enhancement and breeding strategies aimed at improving lodging resistance in japonica rice.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eBADH2\u003c/em\u003e gene encodes the enzyme betaine aldehyde dehydrogenase, which facilitates the conversion of γ-aminobutyraldehyde (GABald) into γ-aminobutyric acid (GABA). This process is integral to plant stress responses and metabolic regulation (Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Mutations that result in the loss of function of \u003cem\u003eBADH2\u003c/em\u003e lead to an accumulation of GABald, which is a precursor in the biosynthesis of 2-acetyl-1-pyrroline (2AP). This accumulation subsequently promotes the increased presence of the aromatic compound 2AP in rice grains (Hui et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, mutations that inactivate \u003cem\u003eBADH2\u003c/em\u003e represent a significant target for molecular breeding strategies aimed at enhancing the fragrance of rice.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eWx\u003c/em\u003e gene encodes granule-bound starch synthase I (GBSSI), which is responsible for catalyzing the synthesis of amylose during the development of the endosperm. The activity of the \u003cem\u003eWx\u003c/em\u003e gene is a determining factor for the amylose content in rice, which in turn affects important eating quality characteristics such as stickiness, hardness, and retrogradation of cooked rice (Zhang et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Variations in the \u003cem\u003eWx\u003c/em\u003e gene can lead to changes in GBSSI activity, resulting in different allelic forms that significantly influence the eating quality of rice (Huang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zeng et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e allele, which is defined by a G to A mutation in the fourth exon (vg0601767613), causes a partial reduction in GBSSI activity, leading to an intermediate amylose content that ranges from 5\u0026ndash;12% (Huang et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a result, the \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e allele has been instrumental in improving the eating quality of japonica rice in Jiangsu Province.\u003c/p\u003e \u003cp\u003eMolecular markers derived from functional variation sites in genes facilitate the swift and effective identification of genotypes linked to desired traits in breeding materials. This approach minimizes the effort required for phenotypic assessment of these traits and enhances the efficiency of selection processes (Kim et al., 2016; Ueda et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eJiangsu Province represents the largest area for japonica rice cultivation in southern China. However, in recent years, extensive lodging in rice production has frequently resulted in decreased yields and compromised quality. The incidence and intensity of rice lodging have notably escalated, largely due to extreme weather events, posing a significant threat to the stability and high productivity of rice cultivation. Incorporating enhanced allelic variations that improve stem strength into traditional japonica rice cultivars in the middle and lower reaches of the Yangtze River represents a promising strategy for developing rice varieties resistant to lodging.\u003c/p\u003e \u003cp\u003eWe evaluated 78 japonica rice germplasm resources from Jiangsu Province for their lodging resistance and yield-related traits, identifying 14 accessions that possess the advantageous \u003cem\u003eSCM3\u003c/em\u003e allele. Notably, Yangchan 93033 and Yangchannuo 1 demonstrated strong stems and high yields, positioning them as valuable germplasm for the development of high-yielding, lodging-resistant rice varieties. To enhance quality traits, we performed a cross between Yangchan 93033 and Wuxiangjing 5245, a premium japonica rice variety known for its favorable alleles associated with fragrance and eating quality. Through multi-generational molecular marker-assisted selection (MAS) and field-based agronomic trait selection, we successfully generated novel japonica rice materials characterized by high yield, lodging resistance, and exceptional eating quality.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCreation of Indel Molecular Markers for Functionally Distinct Regions in \u003cem\u003eSCM3\u003c/em\u003e\u003c/p\u003e \u003cp\u003ePrevious research has established that a 4-bp insertion in the 5' untranslated region (UTR) of the \u003cem\u003eSCM3\u003c/em\u003e gene enhances its expression, leading to an increase in stem diameter (Yano et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This 4-bp variation is therefore recognized as a functional differential site within the \u003cem\u003eSCM3\u003c/em\u003e gene. An analysis using RiceVarMap v2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ricevarmap.ncpgr.cn/\u003c/span\u003e\u003cspan address=\"https://ricevarmap.ncpgr.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) indicated that 97.2% of indica rice varieties contain the 4-bp insertion at the specific locus vg0328428721, whereas only 57.3% of japonica rice varieties exhibit the same insertion. This suggests that the advantageous allele of \u003cem\u003eSCM3\u003c/em\u003e is relatively less common in japonica rice. Thus, promoting the use of this allele in future breeding initiatives could significantly enhance lodging resistance in japonica rice. According to RiceVarMap v2.0, the variation site vg0328428721, situated in the promoter region of the \u003cem\u003eSCM3\u003c/em\u003e gene, displays a 4-bp insertion in the 9311 variety, while this insertion is absent in the Nipponbare (NPB) variety (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). A pair of primers, SCM3_indel_2F/R (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), was designed to flank the variation site vg0328428721. PCR amplification was conducted using genomic DNA from the 9311 and Nipponbare (NPB) varieties as templates. The PCR products were subsequently analyzed through electrophoresis on an 8% polyacrylamide gel. The results revealed distinct target bands of 118 bp and 114 bp for the 9311 and NPB varieties, respectively (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e B). This confirms the presence of the 4-bp insertion in the 9311 variety and its absence in NPB, establishing a reliable molecular marker for differentiating between these two genotypes at the \u003cem\u003eSCM3\u003c/em\u003e locus. Consequently, the two alleles were designated as \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA. The gene structure of \u003cem\u003eSCM3\u003c/em\u003e and the positions of functional variation sites. B. Polyacrylamide gel electrophoresis analysis of SCM3_indel_2.\u003c/p\u003e \u003cp\u003eIdentification of Characteristics for Lodging Resistance and Genotypic Analysis of the \u003cem\u003eSCM3\u003c/em\u003e Gene in 78 Japonica Rice Varieties\u003c/p\u003e \u003cp\u003eA total of 78 rice varieties and advanced lines, either recently approved or currently under evaluation in Jiangsu Province, were randomly selected for allelic genotyping of the \u003cem\u003eSCM3\u003c/em\u003e gene using the SCM3indel-2 molecular marker. The analysis revealed that 14 rice varieties carried the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele, while the remaining 64 varieties exhibited the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). To evaluate the influence of different \u003cem\u003eSCM3\u003c/em\u003e alleles on rice lodging resistance, we investigated six key traits associated with lodging resistance in these 78 rice materials. These traits included plant height (PH), bending strength (BS), lodging index (LI), length of the second internode from the plant root (IL2), culm outer diameter at the cross-section of the second internode from the plant root (CD2), and culm thickness at the cross-section of the second internode from the plant root (CT2) (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA comparative analysis was conducted to evaluate the trait differences between the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e genotypes (Table S3). The average culm diameter (CD2) for rice varieties harboring the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele was recorded at 5.57 mm, while those with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele averaged 5.35 mm, indicating a statistically significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Furthermore, rice varieties with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele demonstrated an average stem wall thickness of 0.84 mm at CT2, in contrast to 0.80 mm for varieties with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele, which also exhibited a significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). The average breaking strength (BS) for rice varieties containing the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele was 21.61 N, significantly exceeding the 18.56 N observed in varieties with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Regarding internode length (IL2), rice varieties with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele averaged 8.27 mm, which was significantly shorter than the 8.71 mm recorded for those with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). However, no significant difference in plant height (PH) was observed between the two genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Lastly, the lodging index (LI) for rice varieties possessing the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele was significantly lower at 40.05 compared to 43.43 for those with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). These results indicate that the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele enhances stem strength and increases resistance to lodging in rice.\u003c/p\u003e \u003cp\u003eTo thoroughly assess the breeding potential of rice germplasm possessing the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele, we performed a statistical analysis on several traits across 78 rice accessions. These traits included the number of grains per panicle (NGP), seed setting rate (SP), thousand grain weight (TGW), grain length (GL), grain width (GW), number of effective panicles (PN), and yield per plant (YP). The analysis revealed that the average NGP for rice germplasm with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele was 212.18, which slightly exceeded the average of 199.75 for germplasm with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Additionally, the average TGW for the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele was 26.30 g, marginally higher than the 25.85 g observed in the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The average GL for germplasm carrying the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele was 7.98 mm, significantly greater than the 7.74 mm for the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, no significant differences were found in SP, GW, and PN between the two alleles (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Furthermore, the average yield per plant for germplasm with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele was 33.49 g, which was significantly higher than the 31.63 g for those with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e allele (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These findings suggest that rice germplasm carrying the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele demonstrates enhanced lodging resistance and yield characteristics. Consequently, through molecular marker-assisted selection, the favorable \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele can be incorporated into japonica rice varieties with desirable traits to develop high-yield, high-quality, lodging-resistant rice cultivars.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis was conducted on various parameters, including the diameter of the second basal internode (A), wall thickness of the second basal internode (B), stem breaking resistance (C), length of the second basal internode (D), plant height (E), and lodging index (F) between the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e genotypes across 78 japonica rice materials. Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the observed differences.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis was conducted on various traits, including the number of grains per panicle (A), 1000-grain weight (B), grain length (C), grain width (D), plant height (E), and yield per plant (F), between the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003eNPB\u003c/em\u003e\u003c/sup\u003e genotypes across 78 japonica rice materials. Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the observed differences.\u003c/p\u003e \u003cp\u003eElite germplasm resources were chosen as parental lines to develop rice varieties characterized by high yield, exceptional quality, and resistance to lodging\u003c/p\u003e \u003cp\u003eAmong the 14 rice germplasm materials possessing the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele, Yangchan 93033 and Yangchannuo 1 demonstrated superior overall performance in terms of F, CD2, TGW, YP, and NGP (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Despite all 14 rice germplasm accessions carrying the advantageous \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele, notable differences in lodging resistance traits were observed among them. This phenotypic variation is likely due to differences in their genetic backgrounds, which may affect the expression or efficacy of the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e allele in providing lodging resistance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis was conducted on various agronomic traits of 14 japonica rice materials possessing the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e genotype. The traits examined included the number of grains per panicle (A), 1000-grain weight (B), grain length (C), grain width (D), plant height (E), and yield per plant (F). For the multiple comparison analysis, a significance level of LSD_0.01 was employed.\u003c/p\u003e \u003cp\u003eWuxiangjing 5245 is recognized as a high-quality aromatic soft rice variety; however, it exhibits lower yield and lodging resistance compared to Yangchan 93033 (Fig. S3). The critical dimensions (CD2) and breaking strength (BS) of Yangchan 93033 were significantly higher than those of Wuxiangjing 5245, indicating its superior lodging resistance (Fig. S3A and S3C). Furthermore, Yangchan 93033 demonstrated markedly elevated net grain production (NGP), thousand grain weight (TGW), and yield potential (YP) relative to Wuxiangjing 5245, highlighting its considerable yield enhancement potential (Fig. S3A and S3C). Therefore, to create new rice varieties that combine high yield, exceptional quality, and improved lodging resistance, we developed hybrid combinations by crossing Yangchan 93033 and Yangchannuo 1 (which serve as donors of the \u003cem\u003eSCM3\u003c/em\u003e gene) with the high-quality and palatable japonica rice variety Wuxiangjing 5245 (Fig. S3G, S3H, and S3L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis was conducted on various parameters between two japonica rice varieties, Yangchan 93033 and Wuxiangjing 5245. The parameters examined included the diameter of the second basal internode (A), wall thickness of the second basal internode (B), stem breaking resistance (C), length of the second basal internode (D), plant height (E), lodging index (F), number of grains per panicle (G), 1000-grain weight (H), grain length (I), grain width (J), number of productive panicles (K), and yield per plant (L). Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the differences observed.\u003c/p\u003e \u003cp\u003eFurther examination of the basal stems of Yangchan 93033 and Wuxiangjing 5245 revealed that the vascular bundle size in Yangchan 93033 was considerably larger than that in Wuxiangjing 5245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCross-sectional microscope images of the second internode at the base for Yangchan 93033 (A) and Wuxiangjing 5245 (B). Cross-sectional paraffin section microscope images of the second internode at the base for Yangchan 93033 (C) and Wuxiangjing 5245 (D).\u003c/p\u003e \u003cp\u003eValidation of the \u003cem\u003eSCM3\u003c/em\u003e Locus for Lodging Resistance Through Bulked Segregant Analysis (BSA).\u003c/p\u003e \u003cp\u003eTo confirm that the \u003cem\u003eSCM3\u003c/em\u003e gene is the primary gene associated with lodging resistance in Yangchannuo 1, we measured the diameter of the second internode at the base of the main stem panicle in 200 individual plants from the F2 population derived from the cross between Yangchannuo 1 and Wuxiangjing 5245. We subsequently selected and pooled leaves from 30 plants exhibiting diameters similar to those of Yangchannuo 1 and 30 plants resembling Wuxiangjing 5245 for resequencing analysis. Both parental lines, Yangchannuo 1 and Wuxiangjing 5245, were also included in the resequencing as controls. Bulked segregant analysis (BSA) identified a significant quantitative trait locus (QTL) for stem strength located within the chromosomal region of 28,400,000 to 28,600,000 on chromosome 3. The \u003cem\u003eSCM3\u003c/em\u003e gene is situated precisely within this interval, thereby confirming that the \u003cem\u003eSCM3\u003c/em\u003e gene is the key gene responsible for the lodging resistance trait observed in Yangchannuo 1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDevelopment and utilization of KASP markers for the \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e genes in breeding programs\u003c/p\u003e \u003cp\u003eResequencing analysis of two rice varieties, Yangchan 93033 and Yangchannuo 1, identified two variant sites in the \u003cem\u003eSCM3\u003c/em\u003e gene when compared to the reference genome NBP. One of these variants is a previously documented functional variant (TGTG/-) located at base pair 28428731 on chromosome 3 of the rice reference genome MSU7.0 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The second variant is a single nucleotide polymorphism (vg0328430214, G/T) situated 1206 bp downstream of the ATG start codon at base pair 28430214 on the same chromosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). An inquiry into the rice variation database indicates that vg0328430214 is found in the 3' UTR region of the \u003cem\u003eSCM3\u003c/em\u003e gene, occurring in only 0.7% of 4726 rice germplasm resources, 0.5% of 2759 indica rice germplasm resources, and 1.1% of 1512 japonica rice germplasm resources (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ricevarmap.ncpgr.cn/vars_info/\u003c/span\u003e\u003cspan address=\"https://ricevarmap.ncpgr.cn/vars_info/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These findings suggest that the vg0328430214 variant in the 3' UTR of the \u003cem\u003eSCM3\u003c/em\u003e gene is highly specific to the natural rice population in Yangchan 93033 and Yangchannuo 1. To improve selection efficiency in breeding for lodging resistance, this specific variant can be developed into a KASP molecular marker (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), providing a valuable tool for selecting the \u003cem\u003eSCM3\u003c/em\u003e gene during the enhancement of lodging resistance traits in rice.\u003c/p\u003e \u003cp\u003eGenome resequencing analysis identified a 7-bp deletion in the second exon of the \u003cem\u003eBADH2\u003c/em\u003e gene (chr08:20380278) and a single nucleotide substitution (G to A) in the fourth exon of the \u003cem\u003eWx\u003c/em\u003e gene (chr06:1767613) in Wuxiangjing 5245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These findings suggest that Wuxiangjing 5245 possesses the advantageous fragrant allele badh2_E2 and the beneficial taste allele \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e when compared to Yangchan 93033. Utilizing the functional variant sites of three genes, \u003cem\u003eSCM3\u003c/em\u003e (vg0328430214, G/T), \u003cem\u003eBADH2\u003c/em\u003e (vg0820380278, CGGGCGC/-------), and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e (vg0601767613, G/A)\u0026mdash;three KASP molecular markers were developed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). These KASP markers facilitate the rapid and efficient identification of the \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e genotypes in individual plants from the Yangchan 93033/Wuxiangjing 5245 F2 population (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSchematic diagrams of the gene structures and locations of variation sites for \u003cem\u003eSCM3\u003c/em\u003e (A), \u003cem\u003eBADH2\u003c/em\u003e (B), and \u003cem\u003eWx\u003c/em\u003e (C). Genotyping results of KASP markers for \u003cem\u003eSCM3\u003c/em\u003e (D), \u003cem\u003eBADH2\u003c/em\u003e (E), and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e (F).\u003c/p\u003e \u003cp\u003eMarker-assisted selection (MAS) was employed to expedite the development of novel rice lines characterized by high yield, enhanced quality, and resistance to lodging\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo create new rice varieties characterized by high yield, enhanced quality, and resistance to lodging, we utilized the elite parental lines Yangchan 93033 and Wuxiangjing 5245 to produce F\u003csub\u003e1\u003c/sub\u003e hybrids through controlled crosses. Following self-pollination and seed harvesting, we established an F\u003csub\u003e2\u003c/sub\u003e segregating population. We cultivated 1,000 F\u003csub\u003e2\u003c/sub\u003e individuals and performed genotyping using three molecular markers: SCM3_k_28430214, BADH2_k_20380278, and Wx_mp_k_1767613, to identify the genotypes associated with the \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e genes, respectively. Upon reaching maturity, we evaluated the agronomic traits of the rice plants and selected individuals that possessed favorable alleles of \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e, exhibiting superior overall traits for seed collection. Ultimately, 156 individuals were selected and planted as F\u003csub\u003e3\u003c/sub\u003e head rows, with each head row comprising three rows of 20 seedlings each.\u003c/p\u003e \u003cp\u003eThe agronomic characteristics of 156 head rows were assessed, resulting in the identification and tagging of 500 plants exhibiting outstanding overall agronomic traits. Leaf samples were gathered for DNA extraction, and the genotypes of \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and Wx-mp were analyzed. In conclusion, 58 plants possessing the advantageous alleles of \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e, along with exceptional comprehensive traits, were selected and progressed to the F\u003csub\u003e4\u003c/sub\u003e generation for additional evaluation.\u003c/p\u003e \u003cp\u003eA total of 58 F\u003csub\u003e4\u003c/sub\u003e head rows were assessed for agronomic characteristics and genotyped for the \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e loci. From this evaluation, 202 plants exhibiting superior overall traits and possessing the advantageous alleles of \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e were selected for advancement to the F\u003csub\u003e5\u003c/sub\u003e generation. To minimize genotyping expenses, only those individuals with heterozygous genotypes from the preceding generation were subjected to genotyping. The 202 F\u003csub\u003e5\u003c/sub\u003e head rows were subsequently evaluated for agronomic traits and genotyped for \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e, leading to the identification of 297 homozygous plants that carried the favorable alleles of \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eBADH2\u003c/em\u003e, and \u003cem\u003eWx\u003c/em\u003e\u003csup\u003e\u003cem\u003emp\u003c/em\u003e\u003c/sup\u003e, which were then advanced to the F\u003csub\u003e6\u003c/sub\u003e generation.\u003c/p\u003e \u003cp\u003eIn the 297 F\u003csub\u003e6\u003c/sub\u003e head rows, agronomic characteristics including heading date, plant height, and panicle type were assessed, leading to the selection of lines exhibiting high uniformity and the absence of segregation. A total of 44 stable lines were identified for the evaluation of lodging resistance, aimed at investigating the impact of \u003cem\u003eSCM3\u003c/em\u003e genotype selection on enhancing lodging resistance in rice (Table S4). The breaking resistance of the 44 F\u003csub\u003e6\u003c/sub\u003e lines was found to be significantly greater than that of Wuxiangjing 5245 (Fig. S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis was conducted on various traits, including the number of productive panicles (A), stem breaking resistance (B), lodging index (C), length of the second basal internode (D), diameter of the second basal internode (E), and wall thickness of the second basal internode (F), among 44 F\u003csub\u003e6\u003c/sub\u003e progeny lines and their parental varieties, Yangchan 93033 and Wuxiangjing 5245. Welch's Two Sample \u003cem\u003et\u003c/em\u003e-test was employed to assess the significance of the observed differences.\u003c/p\u003e \u003cp\u003eAssessment of agronomic characteristics in stable lines\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis was conducted on the number of productive panicles (A), total grains per panicle (B), 1000-grain weight (C), and yield per plant (D) across eight stable elite lines. The least significant difference at the 0.01 level (LSD_0.01) was employed for multiple comparison analysis.\u003c/p\u003e \u003cp\u003eWe identified eight stable lines exhibiting outstanding overall characteristics and superior lodging resistance, based on their lodging resistance performance and other agronomic traits, for a comprehensive evaluation of agronomic traits and an analysis of yield and quality. The number of productive panicles in the lines 24HD134 and 24HD362 was significantly greater than that observed in Wuxiangjing 5245 and Yangchan 93033 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In contrast, the remaining lines exhibited a notable decrease in productive panicle count compared to the two parental varieties (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). All eight stable lines demonstrated a significantly higher number of grains per panicle than Wuxiangjing 5245, with three lines exhibiting values comparable to Yangchan 93033, showing no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, the 1000-grain weight for all eight stable lines was found to be lower than that of Yangchan 93033 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Specifically, the 1000-grain weight of 24HD127 was significantly less than that of both Wuxiangjing 5245 and Yangchan 93033, while the other seven lines did not show significant differences when compared to Wuxiangjing 5245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eAmong the eight lines studied, the yield per plant for 24HD134, 24HD142, 24HD365, and 24HD448 was significantly greater than that of Wuxiangjing 5245, although it did not surpass that of Yangchan 93033 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eIn conclusion, the varieties 24HD134, 24HD142, 24HD365, and 24HD448 showed a notable enhancement in both the number of grains per panicle and the yield per plant when compared to Wuxiangjing 5245, indicating their considerable yield potential.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative analysis was conducted on the brown rice yield (A), milled rice yield (B), sensory evaluation of cooked rice (C), and amylose content in rice flour (D) across eight stable elite lines. The Least Significant Difference (LSD) at the 0.01 level was employed for multiple comparison assessments.\u003c/p\u003e \u003cp\u003eThe brown rice yield of 24HD127 was significantly lower than that of Wuxiangjing 5245, whereas the remaining seven lines did not show any significant differences when compared to Wuxiangjing 5245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Among the eight stable lines, 24HD362 demonstrated the highest milled rice yield, which was significantly greater than that of Wuxiangjing 5245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The cooked rice taste value for 24HD134, 24HD146, and 24HD362 was significantly superior to that of Yangchan 93033 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Additionally, the amylose content in 24HD134, 24HD146, 24HD362, 24HD365, and 24HD448 was lower than that found in Yangchan 93033 (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e7\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA comparative evaluation of stem breaking resistance (A), lodging index (B), diameter of the second basal internode (C), and wall thickness of the second basal internode (D) was conducted among eight stable elite lines. The least significant difference at the 0.01 level (LSD_0.01) was employed for the multiple comparison analysis.\u003c/p\u003e \u003cp\u003eThe breaking strength of the stems (BS) in all eight stable lines was significantly greater than that of Wuxiangjing 5245, yet it did not surpass that of Yangchan 93033 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The lodging index for lines 24HD146, 24HD362, and 24HD448 was significantly higher than that of Yangchan 93033, whereas the lodging index for line 24HD127 was significantly lower than that of Yangchan 93033 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The remaining four lines exhibited no significant differences in lodging index when compared to Yangchan 93033(Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe diameter of the second basal internode in the lines 24HD142, 24HD146, 24HD362, and 24HD448 was significantly greater than that observed in Wuxiangjing 5245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Additionally, the wall thickness of the second basal internode in the lines 24HD112, 24HD142, 24HD146, 24HD362, 24HD365, and 24HD448 was notably thicker compared to Wuxiangjing 5245 (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eThe experimental findings indicate that the 24HD134 line exhibits outstanding overall performance in terms of yield, quality, and resistance to lodging. This positions it as a strong candidate for subsequent production trials as a new line characterized by high yield, superior quality, and enhanced lodging resistance.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs rice variety yields continue to improve, the susceptibility to lodging in high-yielding varieties under extreme climatic conditions has intensified. To expedite the breeding of high-yielding and stable varieties, we systematically assessed lodging resistance traits in 78 japonica rice germplasm resources. Our screening identified 14 rice materials with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e genotype, which exhibited notable lodging resistance characteristics. These materials show considerable breeding potential and can be utilized as important parental resources for the development of lodging-resistant varieties.\u003c/p\u003e \u003cp\u003eA comprehensive investigation and analysis of the agronomic traits of 14 rice varieties with the \u003cem\u003eSCM3\u003c/em\u003e\u003csup\u003e\u003cem\u003e9311\u003c/em\u003e\u003c/sup\u003e genotype revealed that Yangchan 93033 and Yangchannuo 1 possess exceptional overall characteristics. These two varieties are characterized by sturdy stems, strong resistance to bending, a high number of grains per panicle, and elevated single-plant yields, indicating considerable yield potential. Consequently, Yangchan 93033 and Yangchannuo 1 represent valuable germplasm resources for lodging resistance, offering crucial genetic materials for the development of high-yield and stable rice varieties.\u003c/p\u003e \u003cp\u003eThrough Bulked Segregant Analysis (BSA), we have confirmed that the primary gene responsible for stem diameter in Yangchannuo 1 is \u003cem\u003eSCM3\u003c/em\u003e. A comparative analysis of the \u003cem\u003eSCM3\u003c/em\u003e gene sequences from Yangchannuo 1 and Yangchan 93033 identified a unique single nucleotide polymorphism (SNP) site (vg0328430214, G/T) present in both varieties. This SNP is significantly linked to the robust stem characteristic and can be further developed into a Kompetitive Allele-Specific PCR (KASP) molecular marker. The establishment of this marker will enable precise screening for lodging resistance and facilitate molecular marker-assisted breeding, thereby providing essential technical support and genetic resources for the development of high-yield and stable rice varieties.\u003c/p\u003e \u003cp\u003eThe notable enlargement of the cross-sectional area of vascular bundles in rice stems significantly enhances the transport efficiency of non-structural carbohydrates, which in turn promotes dry matter accumulation and ultimately boosts yield (Li et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Specifically, in the case of Yangchan 93033, the cross-sectional area of vascular bundles in the second internode at the base is considerably larger compared to Wuxiangjing 5245. This structural advantage provides Yangchan 93033 with an enhanced capacity for nutrient transport, thereby facilitating dry matter accumulation and leading to a substantial increase in yield.\u003c/p\u003e \u003cp\u003eA progeny population was developed from the cross between Yangchan 93033 and Wuxiangjing 5245, and molecular marker-assisted selection was utilized to evaluate 44 lines from the F\u003csub\u003e6\u003c/sub\u003e generation. These lines demonstrated a notable enhancement in stem breaking resistance and stem diameter when compared to Wuxiangjing 5245, indicating that the \u003cem\u003eSCM3\u003c/em\u003e molecular marker can effectively and swiftly improve lodging resistance in breeding materials. Nonetheless, the breaking resistance of these lines was still significantly inferior to that of Yangchan 93033, which may be attributed to variations in genetic background. Consequently, for future breeding efforts aimed at improving lodging resistance, Yangchan 93033 should be considered as a recurrent parent for backcrossing with high-yield and high-quality materials to fully leverage its advantageous lodging resistance characteristics.\u003c/p\u003e \u003cp\u003eSubsequent evaluation of agronomic traits led to the identification of eight stable lines from the aforementioned population, which were thoroughly assessed for yield and quality. Notably, line 24HD134 demonstrated exceptional performance in terms of yield, flavor quality, and resistance to lodging, resulting in a synergistic enhancement of these traits. This line represents a significant resource for the development of rice varieties that are high-yielding, high-quality, and resistant to lodging. In the future, 24HD134 can be used as a parent line to combine \u003cem\u003eGn1a/OsCKX2\u003c/em\u003e, \u003cem\u003eSTRONG2\u003c/em\u003e and \u003cem\u003eSCM2\u003c/em\u003e through molecular marker-assisted selection, thereby cultivating high-yielding and high-quality rice varieties with improved resistance to lodging.\u003c/p\u003e \u003cp\u003eWhile molecular marker-assisted selection facilitates the swift and effective identification of desired traits, the stabilization of other agronomic characteristics within breeding populations still necessitates several generations of self-fertilization. To expedite the breeding process, future initiatives may explore the use of pollen microspore culture or haploid induction methods to generate doubled haploid lines, which would substantially improve breeding efficiency (Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn August 2024, Yangzhou experienced an average maximum temperature of 36\u0026deg;C, resulting in high-temperature stress during the heading phase around August 18. This stress significantly reduced both the seed setting rate and grain quality in the affected rice lines. The eight stable lines analyzed in this study demonstrated notable variations in their growth duration. To thoroughly assess varietal adaptability, lines that headed around August 18 are recommended for cultivation in northern Jiangsu Province, while those that headed around September 6 are suitable for southern Jiangsu Province. A systematic evaluation of their yield, quality, and lodging resistance will aid in the identification of superior rice lines that are well-suited to different ecological regions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study established a molecular marker based on the functional divergence sites of the \u003cem\u003eSCM3\u003c/em\u003e gene to evaluate lodging resistance in 78 rice germplasm resources. Fourteen accessions with the \u003cem\u003eSCM3⁹\u0026sup3;\u0026sup1;\u0026sup1;\u003c/em\u003e genotype showed exceptional lodging resistance, particularly Yangchan 93033 and Yangchannuo 1, which exhibited strong stems and high grain yield. A KASP marker, SCM3_k_28430214, was confirmed as an effective tool for molecular marker-assisted selection. A hybrid population was created by crossing Yangchan 93033 with Wuxiangjing 5245, leading to the development of a new breeding line, 24HD134, which integrates favorable alleles and demonstrates high yield potential, superior grain quality, and improved lodging resistance.\u003c/p\u003e "},{"header":"Methods","content":" \u003cp\u003eExperimental Materials and Cultivation\u003c/p\u003e \u003cp\u003eThe study utilized 77 rice varieties that have either recently been approved or are currently undergoing trials in Jiangsu Province, along with breeding materials such as Yangchan 93033 and Wuxiangjing 5245. These materials were cultivated at the experimental facility located in Shatou Town, Guangling District, Yangzhou City. All rice varieties were transplanted as individual seedlings, maintaining a plant spacing of 12 cm and a row spacing of 28 cm. Nitrogen fertilizer was applied at a rate of 258.75 kg/ha of pure nitrogen, adhering to a fertilizer application ratio of 4:3:3 for basal, tiller, and panicle fertilizers. Water and fertilizer management, along with pest and disease control, were implemented in accordance with the standards for high-yield rice cultivation.\u003c/p\u003e \u003cp\u003eDevelopment of Functional Markers\u003c/p\u003e \u003cp\u003eA 4-bp variation site (ID: vg0328428721, C/CTGTG) located in the promoter region of the \u003cem\u003eSCM3\u003c/em\u003e gene (\u003cem\u003eLOC_Os03g49880\u003c/em\u003e) was identified from publicly accessible data on the website \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://ricevarmap.ncpgr.cn/\u003c/span\u003e\u003cspan address=\"http://ricevarmap.ncpgr.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Utilizing the flanking sequence information of the vg0328428721 variation site, a pair of primers was designed with Primer Premier 5.0 software. The development and design of the KASP (Kompetitive Allele-Specific PCR) marker were outsourced to Jingtai Biotechnology Co., Ltd., employing the sequences adjacent to the gene variation site.\u003c/p\u003e \u003cp\u003eMolecular marker detection was conducted using standard Polymerase Chain Reaction (PCR) amplification techniques in the laboratory. The reaction mixture, totaling 10 \u0026micro;L, included 1 \u0026micro;L of template DNA (approximately 15 ng \u0026micro;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), 0.4 \u0026micro;L of each forward and reverse primer (10 \u0026micro;mol \u0026micro;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), 5 \u0026micro;L of 2\u0026times;NG PCR MasterMix (Shanghai Huiling Biotechnology Co., Ltd., NG001M), and 3.2 \u0026micro;L of sterile double-distilled water. The amplification process was performed in a PCR machine under the following conditions: (1) initial denaturation at 95\u0026deg;C for 5 minutes; (2) 35 cycles consisting of denaturation at 95\u0026deg;C for 30 seconds, annealing at 55\u0026deg;C for 30 seconds, and extension at 72\u0026deg;C for 30 seconds; and (3) a final extension at 72\u0026deg;C for 10 minutes. The resulting PCR products were analyzed by electrophoresis on an 8% polyacrylamide gel.\u003c/p\u003e \u003cp\u003ePCR amplification of the KASP marker was conducted utilizing the PARMS mix from Wuhan Jingtai Biotechnology. The reaction system, totaling 10 \u0026micro;L, comprised approximately 50 ng of template DNA, 0.15 \u0026micro;L each of two allele-specific primers (10 \u0026micro;mol L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), 5 \u0026micro;L of 2\u0026times;PARMS master mix (Wuhan Jingtai Biotechnology), and sterile double-distilled water to achieve the final volume. The amplification process was executed in a PCR instrument under the following conditions: (1) initial denaturation at 94\u0026deg;C for 15 minutes; (2) 10 cycles of denaturation at 94\u0026deg;C for 20 seconds, followed by annealing at a temperature decreasing from 65\u0026deg;C to 57\u0026deg;C (decreasing by 0.8\u0026deg;C per cycle) for 60 seconds; and (3) 32 cycles of denaturation at 94\u0026deg;C for 20 seconds and annealing at 57\u0026deg;C for 60 seconds. Upon completion of the PCR, fluorescence signals were detected using a TECAN Infinite M1000 microplate reader. These signals were subsequently analyzed and transformed into clear and intuitive genotyping plots via the online software snpdecoder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.snpway.com/snpdecoder/\u003c/span\u003e\u003cspan address=\"http://www.snpway.com/snpdecoder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The genotyping results were generated based on the observed color differences.\u003c/p\u003e \u003cp\u003eEvaluation of Stem Robustness in Rice Cultivars\u003c/p\u003e \u003cp\u003eThe heading date of the test materials was meticulously documented. At 25 days post-heading, three plants from each material were chosen to assess plant height (PH) and the number of panicles per plant (PN). The main stems, along with the fibrous roots, were severed and promptly placed in water to avoid dehydration before being transported to the laboratory for the measurement of stem traits.\u003c/p\u003e \u003cp\u003eThe collected stems were positioned on the compression force gauge holder of a plant stem strength tester (Zhejiang Top Instrument Co., Ltd., model: YYD-1A), with the distance between the two support points adjusted to 9 cm. The handle of the force gauge was pressed downward until the stem fractured, and the maximum pressure recorded was designated as the bending stress (BS). A ruler and balance were utilized to measure the length from the fractured section of the basal stem to the apex of the panicle (Stem Length, SL) and to determine the fresh weight (FW). The lodging index (LI) was calculated using the formula: LI\u0026thinsp;=\u0026thinsp;SL \u0026times; FW / (BS \u0026times; 9) / 4.\u003c/p\u003e \u003cp\u003eThe length of the second internode from the plant root (IL2) was measured with a ruler. The outer diameters of the second internode, both short-axis and long-axis (excluding the leaf sheath), were measured using a vernier caliper, and the average was recorded as the culm outer diameter at the cross-section of the second internode (CD2). Likewise, the thicknesses of the cross-section of the second internode (excluding the leaf sheath) were measured for both short-axis and long-axis using a vernier caliper, with the average value documented as the culm thickness at the cross-section of the second internode (CT2).\u003c/p\u003e \u003cp\u003eAssessment of Rice Crop Production\u003c/p\u003e \u003cp\u003eUpon reaching full maturity, ten main stem panicles were randomly selected from healthy plants located in the central section of each row. These harvested panicles were placed in mesh bags for drying. Following the drying process, the grains were threshed, and both unfilled and filled grains were counted separately. The total grain count per panicle (NGP) was determined by adding the numbers of unfilled and filled grains. The seed setting rate (SP) was calculated as the ratio of filled grains to the total grain count per panicle. The filled grains from each main stem panicle were preserved and subsequently analyzed using a grain analyzer to assess characteristics such as grain length, grain width, and the weight of 1000 grains.\u003c/p\u003e \u003cp\u003eFurthermore, ten healthy plants from each line were randomly chosen, harvested, and placed in mesh bags for the drying process. The dried plants were then threshed using a small thresher, and the grains from each plant were weighed individually. The average grain weight per plant was computed and documented as the single plant yield (YP) for each line.\u003c/p\u003e \u003cp\u003eImaging Cross-Sections of the Rice Stem Base\u003c/p\u003e \u003cp\u003eAt 30 days after heading, representative main tillers were collected and the leaf sheaths were carefully removed. The basal second internode was excised and immediately fixed in FAA solution (70% ethanol 90 mL, glacial acetic acid 5 mL, formaldehyde 5 mL) for 24 h to preserve cellular integrity. Samples were then softened in an appropriate maceration solution until fully pliable, followed by dehydration and paraffin embedding. Serial sections were cut and stained in safranin for 1\u0026ndash;2 h. Excess stain was removed by rinsing in tap water, and sections were briefly decolorized (3\u0026ndash;8 s each) through a graded ethanol series (50%, 70%, 80%). Thereafter, sections were counterstained in fast green for 30\u0026ndash;60 s, dehydrated through three changes of absolute ethanol, and cleared in fresh xylene for 5 min. Finally, slides were mounted with neutral gum for long-term preservation. Observations and photomicrographs were obtained under a Leica S8 APO stereomicroscope, and morphological measurements of vascular bundle number, area, and perimeter were carried out on the acquired images.\u003c/p\u003e \u003cp\u003eAssessment of Rice Processing, Visual Characteristics, and Flavor Quality\u003c/p\u003e \u003cp\u003eDetermination of Brown Rice Yield: Following the harvesting process, the rice undergoes threshing and sun-drying before being stored in a controlled indoor environment for a duration of three months. A sample weighing 150 grams is extracted from each batch. The brown rice is produced by milling the sample with a rice husker, and the weight of the resulting brown rice is recorded to facilitate the calculation of the brown rice yield. This procedure is replicated three times, ensuring that the discrepancy between the first and third measurements remains within 2%. The final result is derived from the average of the three measurements. The brown rice yield is calculated using the formula:\u003c/p\u003e \u003cp\u003eBrown Rice Yield = (Weight of Brown Rice (g) / 150g) \u0026times; 100%\u003c/p\u003e \u003cp\u003eDetermination of Milled Rice Rate: The brown rice produced in the preceding step undergoes additional milling with a rice polisher, and the weight of the resulting milled rice is documented. This procedure is conducted three times, ensuring that the discrepancy between the first and third measurements does not exceed 1%. The final result is derived from the average of these three measurements. The milled rice rate is computed using the following formula:\u003c/p\u003e \u003cp\u003eMilled Rice Rate = (Weight of Milled Rice (g) / 150g) \u0026times; 100%\u003c/p\u003e \u003cp\u003eDetermination of Transparency, Chalkiness, and Chalky Grain Rate: A random sample of 100 intact milled rice grains is uniformly distributed on the scanning plate of the rice appearance quality analyzer. The Wanshen SC-E Rice Appearance Quality Analyzer software is utilized to assess transparency, chalkiness, and the chalky grain rate. Each measurement is conducted in triplicate, ensuring that the error for transparency remains within 2%, for chalkiness within 10%, and for the chalky grain rate within 5%. The final result is derived from the average of the three measurements.\u003c/p\u003e \u003cp\u003eTaste Quality Assessment: A 30g sample of intact milled rice is placed in a stainless steel container, rinsed, and subsequently combined with water at a ratio of 1:1.3. The container is sealed using a rubber ring and filter paper, and the mixture is soaked for 30 minutes. Following this, the rice is steamed for 30 minutes, allowed to rest for 10 minutes, and then cooled for 20 minutes utilizing a forced air cooling system. After resting at room temperature for 90 minutes, an 8g sample of the cooked rice is extracted, and taste quality along with related parameters is evaluated using a rice taste analyzer (STA1A) manufactured by Satake Corporation. This procedure is repeated three times, and the average value is recorded as the final result.\u003c/p\u003e \u003cp\u003eAssessment of Amylose Levels in Rice\u003c/p\u003e \u003cp\u003eDetermination of Amylose Content: Weigh 0.05 g of rice flour that has been sieved through a 100-mesh screen and transfer it into a 50 mL digestion tube. Add 0.5 mL of 95% ethanol and 4.5 mL of 1 mol/L NaOH to the tube. Heat the mixture in a boiling water bath for 10 minutes, then allow it to cool to room temperature. Finally, dilute the solution with distilled water to achieve a final volume of 50 mL, thereby preparing the sample solution.\u003c/p\u003e \u003cp\u003eTransfer 2.5 mL of the sample solution into a 50 mL digestion tube. Acidify the mixture by adding 0.5 mL of 1 mol/L acetic acid solution, followed by the addition of 0.75 mL of iodine solution. Mix thoroughly and dilute with distilled water to achieve a final volume of 50 mL. Let the solution sit for 20 minutes.\u003c/p\u003e \u003cp\u003eAdjust the spectrophotometer to a wavelength of 620 nm and calibrate it using a blank solution. Subsequently, measure the absorbance of the sample. Repeat this procedure with a standard sample that has a known amylose content to obtain its absorbance and create a standard curve. The amylose content of the sample is then determined using this standard curve.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eData processing and statistical analyses were conducted using Microsoft Excel 2013. The \u003cem\u003et\u003c/em\u003e.test function from the tidyr R package was employed to assess significant differences between groups.\u003c/p\u003e \u003cp\u003eThe analysis of variance (ANOVA) for multiple comparisons was conducted using the AOV analysis module of the QTL IciMapping software, which can be accessed at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://isbreeding.caas.cn/rj/index.htm\u003c/span\u003e\u003cspan address=\"https://isbreeding.caas.cn/rj/index.htm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Project funded by National Key Research and Development Program of China (2022YFD1200104), China Postdoctoral Science Foundation (2021M702767), and Revitalization of Seed Industry in Jiangsu Province: JBGS Project（JBGS［2021］036）.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e’\u003c/strong\u003e\u003cstrong\u003econtributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHJL, ZJY, LGH and ZY conceived and designed the experiments. HJL, ZY, ZZX, MP, ZX, ZNB and ZJY performed the experiments and analyzed the data. HJL, ZY and ZJY was responsible for material plant and field management. HJL wrote the manuscript. WHY, ZY, LGH and ZHC revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAsano K, Takashi T, Miura K, Qian Q, Kitano H, Matsuoka M, Ashikari M (2007) Genetic and molecular analysis of utility of sd1 alleles in rice breeding. \u003cem\u003eBreeding Science\u003c/em\u003e, \u003cem\u003e57\u003c/em\u003e(1), 53\u0026ndash;58. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1270/jsbbs.57.53\u003c/span\u003e\u003cspan address=\"10.1270/jsbbs.57.53\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003eChen, J., Li, S., Zhou, L., Zha, W., Xu, H., \u0026amp; Liu, K. (2024). 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J Integr Plant Biol. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jipb.13011\u003c/span\u003e\u003cspan address=\"10.1111/jipb.13011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"MAS, SCM3, gene pyramiding, rice lodging resistance, rice yield, rice quality","lastPublishedDoi":"10.21203/rs.3.rs-6845239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6845239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eUtilizing the functional divergence sites of \u003cem\u003eSCM3\u003c/em\u003e, we established a molecular marker and performed \u003cem\u003eSCM3\u003c/em\u003e genotyping along with an evaluation of lodging resistance traits in 78 rice germplasm resources. Fourteen accessions with the \u003cem\u003eSCM3⁹\u0026sup3;\u0026sup1;\u0026sup1;\u003c/em\u003e genotype demonstrated exceptional lodging resistance. In particular, Yangchan 93033 and Yangchannuo 1 exhibited strong stems, high bending resistance, and a significant number of grains per panicle, indicating their potential as elite donor parents for breeding lodging-resistant rice. Resequencing analysis revealed a rare variant site in the \u003cem\u003eSCM3\u003c/em\u003e gene of both Yangchan 93033 and Yangchannuo 1, and the derived KASP marker SCM3_k_28430214 was confirmed as an effective tool for molecular marker-assisted selection. A hybrid population was created by crossing Yangchan 93033 with Wuxiangjing 5245, a high-quality japonica cultivar known for its superior taste, to form a segregating breeding population. KASP markers targeting \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eWx\u003c/em\u003e, and \u003cem\u003eBADH2\u003c/em\u003e were utilized for genotypic screening of the progeny. Among 44 F₆ stable lines carrying \u003cem\u003eSCM3⁹\u0026sup3;\u0026sup1;\u0026sup1;\u003c/em\u003e, there was a significant enhancement in lodging resistance compared to Wuxiangjing 5245, validating the effective selection capability of the SCM3_k_28430214 marker in improving lodging resistance. Through molecular marker-assisted selection, we rapidly developed a new breeding line, 24HD134, which integrates favorable alleles of \u003cem\u003eSCM3\u003c/em\u003e, \u003cem\u003eWx\u003c/em\u003e, and \u003cem\u003eBADH2\u003c/em\u003e. This line demonstrates high yield potential, superior grain quality, and improved lodging resistance, offering valuable genetic resources for rice breeding programs.\u003c/p\u003e","manuscriptTitle":"Molecular Marker-Assisted Pyramiding of SCM3, Wx, and BADH2 Genes for the Development of High-Yield, Superior-Quality, and Lodging-Resistant Rice Varieties","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-17 12:56:38","doi":"10.21203/rs.3.rs-6845239/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ae4373d-70c8-4053-91b0-6e827909af37","owner":[],"postedDate":"June 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-01T06:38:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-17 12:56:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6845239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6845239","identity":"rs-6845239","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}